Compositions and methods for treating immune disorders using immune modulating lactococcus bacteria strains

ABSTRACT

Provided herein are methods and compositions related to immune modulating Lactococcus strains useful as therapeutic agents. In certain embodiments, provided herein are methods of treating an immune disorder in a subject comprising administering to the subject a bacterial composition comprising Lactococcus lactis cremoris Strain A (ATCC Deposit Number PTA-125368).

RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 16/192,172, filed Nov. 15, 2018, which claims the benefit ofpriority to U.S. Provisional Patent Applications having Ser. No.62/586,604, filed Nov. 15, 2017, 62/660,693, filed Apr. 20, 2018,62/661,459, filed Apr. 23, 2018, and 62/721,941 filed Aug. 23, 2018, thecontents of each of which are hereby incorporated herein by reference intheir entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 2, 2019, isnamed ETB-01701_SL.txt and is 482,487 bytes in size.

SUMMARY

In certain aspects, provided herein are methods and compositions (e.g.,bacterial compositions, pharmaceutical compositions) related to thetreatment and/or prevention of disease (e.g., cancer, autoimmunedisease, inflammatory disease, metabolic disease), in a subject (e.g., ahuman subject) comprising administering a bacterial compositioncomprising Lactococcus bacteria and/or a product of such bacteria (e.g.,extracellular vesicles (EVs) and/or pharmaceutically active biomasses(PhABs)). In certain aspects, provided herein are methods andcompositions related to the treatment and/or prevention of an immunedisorder in a subject (e.g., a human subject) comprising administering abacterial (pharmaceutical) composition comprising immune modulatingLactococcus bacteria disclosed herein and/or a product of immunemodulating Lactococcus bacteria disclosed herein (e.g., extracellularvesicles (EVs) and/or pharmaceutically active biomasses (PhABs)). Alsoprovided herein are methods of making and/or identifying such abacterium and/or bacterial product. In some embodiments, provided hereare bioreactors comprising Lactococcus bacteria disclosed herein.

In certain embodiments, provided herein are immune modulatingLactococcus bacteria. In some embodiments the immune modulatingLactococcus bacteria is an immune modulating strain of Lactococcuslactis cremoris. In certain embodiments the immune modulatingLactococcus strain is Lactococcus lactis cremoris Strain A (ATCC DepositNumber PTA-125368). In some embodiments, the immune modulating bacteriais a strain of Lactococcus bacteria (e.g., a strain of Lactococcuslactis cremoris) comprising a protein listed in Table 1 and/or a geneencoding a protein listed in Table 1. In some embodiments, the immunemodulating bacteria is a strain of Lactococcus bacteria (e.g., a strainof Lactococcus lactis cremoris) comprising a membrane associated proteinlisted in Table 2 and/or a gene encoding a membrane associated proteinlisted in Table 2. In some embodiments, the immune modulating bacteriais a strain of Lactococcus bacteria (e.g., a strain of Lactococcuslactis cremoris) free or substantially free of a protein listed in Table3 and/or a gene encoding a protein listed in Table 3. In someembodiments, the immune modulating bacteria is a strain of Lactococcusbacteria (e.g., a strain of Lactococcus lactis cremoris) free orsubstantially free of an exopolysaccharide (EPS) synthesis proteinlisted in Table 4 and/or a gene encoding an EPS synthesis protein listedin Table 4. In some embodiments, the immune modulating bacteria is astrain of Lactococcus bacteria (e.g., a strain of Lactococcus lactiscremoris) free or substantially free of an EPS synthesized in whole orin part by a protein listed in Table 4. In some embodiments, the immunemodulating bacteria is a strain of Lactococcus bacteria (e.g., a strainof Lactococcus lactis cremoris) free or substantially free of EPS. Insome embodiments, the bacterial compositions provided herein comprise animmune modulating Lactococcus strain provided herein. In someembodiments, the immune modulating bacteria is a strain of Lactococcusbacteria (e.g., a strain of Lactococcus lactis cremoris) free orsubstantially free of a protein listed in Table 5 and/or a gene encodinga protein listed in Table 5. In some embodiments, the immune modulatingbacteria is a strain of Lactococcus bacteria (e.g., a strain ofLactococcus lactis cremoris) comprising a protein listed in Table 6and/or a gene encoding a protein listed in Table 6.

In some embodiments, provided herein are PhABs made from and/orcomprising an immune modulating Lactococcus strain provided herein. Insome embodiments, the PhABs comprise whole cells, fractions of cells,supernatant from fermentation, fractions of supernatant and/orextracellular vesicles made immune modulating bacteria described herein.In some embodiments, the bacterial compositions provided herein comprisean immune modulating Lactococcus strain PhAB provided herein.

In some embodiments, provided herein are EVs produced by and/orgenerated from and/or isolated from an immune modulating Lactococcusstrain provided herein. In some embodiments, the bacterial compositionscomprise both immune modulating Lactococcus strain EVs and whole immunemodulating Lactococcus strain bacteria (e.g., live bacteria, killedbacteria, attenuated bacteria). In certain embodiments, provided hereinare bacterial compositions comprising immune modulating Lactococcusstrain bacteria (e.g., Lactococcus lactis cremoris Strain A) in theabsence of immune modulating Lactococcus strain EVs. In someembodiments, the pharmaceutical compositions comprise immune modulatingLactococcus strain EVs in the absence of immune modulating Lactococcusstrain bacteria.

In some embodiments, the immune modulating Lactococcus strain comprisesat least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity (e.g., at least 99.5% sequence identity, at least99.6% sequence identity, at least 99.7% sequence identity, at least99.8% sequence identity, at least 99.9% sequence identity) to thenucleotide sequence (e.g., genomic sequence, 16S sequence, CRISPRsequence) of the Lactococcus lactis cremoris Strain A. In someembodiments, the administration of the bacterial composition treats theimmune disorder in the subject. In some embodiments, the immune disorderis an autoimmune disease. In some embodiments, the immune disorder is aninflammatory disease. In some embodiments, the immune disorder is anallergy.

In certain embodiments, provided herein are methods of treating asubject who has an immune disorder (e.g., an autoimmune disease, aninflammatory disease, an allergy), comprising administering to thesubject a bacterial composition comprising immune modulating Lactococcusstrain bacteria provided herein (e.g., a killed bacterium, a livebacterium, a pharmaceutically active biomass and/or an attenuatedbacterium). In some embodiments, immune modulating Lactococcus strain isLactococcus lactis cremoris Strain A (ATCC Deposit Number PTA-125368).In some embodiments, immune modulating Lactococcus strain is a straincomprising at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% sequence identity (e.g., genomic sequence identity, 16Ssequence identity, CRISPR sequence identity) (e.g., at least 99.5%sequence identity, at least 99.6% sequence identity, at least 99.7%sequence identity, at least 99.8% sequence identity, at least 99.9%sequence identity) to the corresponding nucleotide sequence of theLactococcus lactis cremoris Strain A (ATCC Deposit Number PTA-125368).In some embodiments, at least 50%, 60%, 70%, 80%, 85%, 90%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the bacteria in thebacterial composition are the immune modulating Lactococcus strain. Insome embodiments, all or substantially all of the bacteria in thebacterial formulation are the immune modulating Lactococcus strain. Insome embodiments, the bacterial formulation comprises at least 1×10⁵,5×10⁵, 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶,1×10⁷, 2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸,2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸ or 1×10⁹ colonyforming units of the immune modulating Lactococcus strain. In someembodiments, the bacterial composition comprises EVs and/or PhABs (e.g.,whole cells, fractions of cells, supernatant from fermentation,fractions of supernatant and/or extracellular vesicles) made from theimmune modulating Lactococcus strain.

In certain embodiments, provided herein are bacterial compositionscomprising an immune modulating Lactococcus strain provided herein(e.g., a killed bacterium, a live bacterium, a pharmaceutically activebiomass and/or an attenuated bacterium). In some embodiments, immunemodulating Lactococcus strain is Lactococcus lactis cremoris Strain A(ATCC Deposit Number PTA-125368). In some embodiments, immune modulatingLactococcus strain is a strain comprising at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% sequence identity (e.g.,genomic sequence identity, 16S sequence identity, CRISPR sequenceidentity) (e.g., at least 99.5% sequence identity, at least 99.6%sequence identity, at least 99.7% sequence identity, at least 99.8%sequence identity, at least 99.9% sequence identity) to thecorresponding nucleotide sequence of the Lactococcus lactis cremorisStrain A (ATCC Deposit Number PTA-125368). In some embodiments, at least50%, 60%, 70%, 80%, 85%, 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% of the bacteria in the bacterial composition are the immunemodulating Lactococcus strain. In some embodiments, all or substantiallyall of the bacteria in the bacterial formulation are the immunemodulating Lactococcus strain. In some embodiments, the bacterialformulation comprises at least 1×10⁵, 5×10⁵, 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶,5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷,6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸,7×10⁸, 8×10⁸, 9×10⁸ or 1×10⁹ colony forming units of the immunemodulating Lactococcus strain. In some embodiments, the bacterialcomposition comprises EVs and/or PhABs (e.g., whole cells, fractions ofcells, supernatant from fermentation, fractions of supernatant and/orextracellular vesicles) made from the immune modulating Lactococcusstrain.

In certain embodiments, the bacterial compositions provided hereincomprise a specific ratio of immune modulating Lactococcus strainbacteria to immune modulating Lactococcus strain EV particles. Forexample, in some embodiments, the bacterial composition comprises atleast 1 immune modulating Lactococcus strain bacterium for every 1, 1.1,1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6,5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1,7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6,8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 11,12, 13, 14, 15, 16, 17, 18. 19, 20, 21, 22, 23, 24, 25, 26, 27, 28. 29,30, 31, 32, 33, 34, 35, 36, 37, 38. 39, 40, 41, 42, 43, 44, 45, 46, 47,48. 49, 50, 51, 52, 53, 54, 55, 56, 57, 58. 59, 60, 61, 62, 63, 64, 65,66, 67, 68. 69, 70, 71, 72, 73, 74, 75, 76, 77, 78. 79, 80, 81, 82, 83,84, 85, 86, 87, 88. 89, 90, 91, 92, 93, 94, 95, 96, 97, 98. 99, 100,150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800,850, 900, 950, 1×10³, 2×10³, 3×10³, 4×10³, 5×10³, 6×10³, 7×10³, 8×10³,9×10³, 1×10⁴, 2×10⁴, 3×10⁴, 4×10⁴, 5×10⁴, 6×10⁴, 7×10⁴, 8×10⁴, 9×10⁴,1×10⁵, 2×10⁵, 3×10⁵, 4×10⁵, 5×10⁵, 6×10⁵, 7×10⁵, 8×10⁵, 9×10⁵, 1×10⁶,2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷,3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸,4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹,5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰,5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹,5×10¹¹, 6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹, and/or 1×10¹² immune modulatingLactococcus strain EV particles. In some embodiments, the bacterialcomposition comprises about 1 immune modulating Lactococcus strainbacterium for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5,6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8,8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5,9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14, 15, 16, 17, 18. 19, 20, 21, 22,23, 24, 25, 26, 27, 28. 29, 30, 31, 32, 33, 34, 35, 36, 37, 38. 39, 40,41, 42, 43, 44, 45, 46, 47, 48. 49, 50, 51, 52, 53, 54, 55, 56, 57, 58.59, 60, 61, 62, 63, 64, 65, 66, 67, 68. 69, 70, 71, 72, 73, 74, 75, 76,77, 78. 79, 80, 81, 82, 83, 84, 85, 86, 87, 88. 89, 90, 91, 92, 93, 94,95, 96, 97, 98. 99, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550,600, 650, 700, 750, 800, 850, 900, 950, 1×10³, 2×10³, 3×10³, 4×10³,5×10³, 6×10³, 7×10³, 8×10³, 9×10³, 1×10⁴, 2×10⁴, 3×10⁴, 4×10⁴, 5×10⁴,6×10⁴, 7×10⁴, 8×10⁴, 9×10⁴, 1×10⁵, 2×10⁵, 3×10⁵, 4×10⁵, 5×10⁵, 6×10⁵,7×10⁵, 8×10⁵, 9×10⁵, 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶,8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷,9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸,1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰,2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹,2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹, 6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹, and/or1×10¹² immune modulating Lactococcus strain EV particles. In someembodiments, the bacterial composition comprises no more than 1Lactococcus lactis cremoris bacterium for every 1, 1.1, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9,3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4,4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9,6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4,7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9,9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14, 15,16, 17, 18. 19, 20, 21, 22, 23, 24, 25, 26, 27, 28. 29, 30, 31, 32, 33,34, 35, 36, 37, 38. 39, 40, 41, 42, 43, 44, 45, 46, 47, 48. 49, 50, 51,52, 53, 54, 55, 56, 57, 58. 59, 60, 61, 62, 63, 64, 65, 66, 67, 68. 69,70, 71, 72, 73, 74, 75, 76, 77, 78. 79, 80, 81, 82, 83, 84, 85, 86, 87,88. 89, 90, 91, 92, 93, 94, 95, 96, 97, 98. 99, 100, 150, 200, 250, 300,350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1×10³,2×10³, 3×10³, 4×10³, 5×10³, 6×10³, 7×10³, 8×10³, 9×10³, 1×10⁴, 2×10⁴,3×10⁴, 4×10⁴, 5×10⁴, 6×10⁴, 7×10⁴, 8×10⁴, 9×10⁴, 1×10⁵, 2×10⁵, 3×10⁵,4×10⁵, 5×10⁵, 6×10⁵, 7×10⁵, 8×10⁵, 9×10⁵, 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶,5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷,6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸,7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹,8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 7×10¹⁰,8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹, 6×10¹¹, 7×10¹¹,8×10¹¹, 9×10¹¹, and/or 1×10¹² immune modulating Lactococcus strain EVparticles. In some embodiments, the bacterial composition comprises atleast 1 immune modulating Lactococcus strain EV particle for every 1,1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5,2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4,4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5,5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7,7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5,8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10,11, 12, 13, 14, 15, 16, 17, 18. 19, 20, 21, 22, 23, 24, 25, 26, 27, 28.29, 30, 31, 32, 33, 34, 35, 36, 37, 38. 39, 40, 41, 42, 43, 44, 45, 46,47, 48. 49, 50, 51, 52, 53, 54, 55, 56, 57, 58. 59, 60, 61, 62, 63, 64,65, 66, 67, 68. 69, 70, 71, 72, 73, 74, 75, 76, 77, 78. 79, 80, 81, 82,83, 84, 85, 86, 87, 88. 89, 90, 91, 92, 93, 94, 95, 96, 97, 98. 99, 100,150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800,850, 900, 950, 1×10³, 2×10³, 3×10³, 4×10³, 5×10³, 6×10³, 7×10³, 8×10³,9×10³, 1×10⁴, 2×10⁴, 3×10⁴, 4×10⁴, 5×10⁴, 6×10⁴, 7×10⁴, 8×10⁴, 9×10⁴,1×10⁵, 2×10⁵, 3×10⁵, 4×10⁵, 5×10⁵, 6×10⁵, 7×10⁵, 8×10⁵, 9×10⁵, 1×10⁶,2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷,3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸,4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹,5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰,5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹,5×10¹¹, 6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹, and/or 1×10¹² immune modulatingLactococcus strain bacteria. In some embodiments, the bacterialcomposition comprises about 1 immune modulating Lactococcus strain EVparticle for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2,2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5,6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8,8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5,9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14, 15, 16, 17, 18. 19, 20, 21, 22,23, 24, 25, 26, 27, 28. 29, 30, 31, 32, 33, 34, 35, 36, 37, 38. 39, 40,41, 42, 43, 44, 45, 46, 47, 48. 49, 50, 51, 52, 53, 54, 55, 56, 57, 58.59, 60, 61, 62, 63, 64, 65, 66, 67, 68. 69, 70, 71, 72, 73, 74, 75, 76,77, 78. 79, 80, 81, 82, 83, 84, 85, 86, 87, 88. 89, 90, 91, 92, 93, 94,95, 96, 97, 98. 99, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550,600, 650, 700, 750, 800, 850, 900, 950, 1×10³, 2×10³, 3×10³, 4×10³,5×10³, 6×10³, 7×10³, 8×10³, 9×10³, 1×10⁴, 2×10⁴, 3×10⁴, 4×10⁴, 5×10⁴,6×10⁴, 7×10⁴, 8×10⁴, 9×10⁴, 1×10⁵, 2×10⁵, 3×10⁵, 4×10⁵, 5×10⁵, 6×10⁵,7×10⁵, 8×10⁵, 9×10⁵, 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶,8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷,9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸,1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰,2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹,2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹, 6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹, and/or1×10¹² immune modulating Lactococcus strain bacteria. In someembodiments, the bacterial composition comprises no more than 1 immunemodulating Lactococcus strain EV particle for every 1, 1.1, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3,4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8,5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3,7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8,8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14,15, 16, 17, 18. 19, 20, 21, 22, 23, 24, 25, 26, 27, 28. 29, 30, 31, 32,33, 34, 35, 36, 37, 38. 39, 40, 41, 42, 43, 44, 45, 46, 47, 48. 49, 50,51, 52, 53, 54, 55, 56, 57, 58. 59, 60, 61, 62, 63, 64, 65, 66, 67, 68.69, 70, 71, 72, 73, 74, 75, 76, 77, 78. 79, 80, 81, 82, 83, 84, 85, 86,87, 88. 89, 90, 91, 92, 93, 94, 95, 96, 97, 98. 99, 100, 150, 200, 250,300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950,1×10³, 2×10³, 3×10³, 4×10³, 5×10³, 6×10³, 7×10³, 8×10³, 9×10³, 1×10⁴,2×10⁴, 3×10⁴, 4×10⁴, 5×10⁴, 6×10⁴, 7×10⁴, 8×10⁴, 9×10⁴, 1×10⁵, 2×10⁵,3×10⁵, 4×10⁵, 5×10⁵, 6×10⁵, 7×10⁵, 8×10⁵, 9×10⁵, 1×10⁶, 2×10⁶, 3×10⁶,4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, 4×10⁷,5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸,6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹,7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰, 6×10¹⁰,7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹, 6×10¹¹,7×10¹¹, 8×10¹¹, 9×10¹¹, and/or 1×10¹² immune modulating Lactococcusstrain bacteria.

In some embodiments, the bacterial composition is administered orally,intravenously, intratumorally, or subcutaneously. In some embodiments,the bacterial composition is administered in 2 or more (e.g., 3 or more,4 or more or 5 or more doses). In some embodiments, the administrationto the subject of the two or more doses are separated by at least 1hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16hours, 17 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or 21 days.In some embodiments, a second bacterium is administered as part of anecological consortium.

In some embodiments, the subject has mild to moderate atopic dermatitis.In some embodiments, the subject has mild atopic dermatitis. In someembodiments, the subject has moderate atopic dermatitis.

In some embodiments, the subject has mild to moderate psoriasis. In someembodiments, the subject has mild psoriasis. In some embodiments thesubject has moderate psoriasis.

In some embodiments, the subject is administered a daily dose of betweenabout 66 mg and about 3.3 g of an immune modulating Lactococcus strainprovided herein (e.g., Lactococcus lactis cremoris Strain A (ATCCDeposit Number PTA-125368) or a strain comprising at least 99% sequenceidentity (e.g., genomic sequence identity, 16S sequence identity, CRISPRsequence identity) (e.g., at least 99.5% sequence identity, at least99.6% sequence identity, at least 99.7% sequence identity, at least99.8% sequence identity, at least 99.9% sequence identity) to thenucleotide sequence of the Lactococcus lactis cremoris Strain A (ATCCDeposit Number PTA-125368)). In some embodiments, the subject isadministered a daily dose of about 66 mg of an immune modulatingLactococcus strain provided herein. In some embodiments, the subject isadministered a daily dose of about 660 mg of an immune modulatingLactococcus strain provided herein. In some embodiments, the subject isadministered a daily dose of about 3.3 g of an immune modulatingLactococcus strain provided herein. In some embodiments, the daily doseis formulated in a capsule. In some embodiments, the subject isadministered the dose of an immune modulating Lactococcus strainprovided herein for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days.

In some embodiments, the subject has a body mass index of 18 kg/m2 to 35kg/m2. In some embodiments, the subject has a confirmed diagnosis ofmild to moderate plaque-type psoriasis for at least 6 months involving≤5% of body surface area (BSA) (excluding the scalp). In someembodiments, the subject ha a minimum of 2 psoriatic lesions. In someembodiments, the subject has mild to moderate atopic dermatitis with aminimum of 3 to a maximum of 15% BSA involvement. In some embodiments,the subject has had a confirmed diagnosis of mild to moderate atopicdermatitis for at least 6 months with an IGA score of 2 or 3. In someembodiments, the subject has at least 2 atopic dermatitis lesions.

In some embodiments, the subject is not pregnant. In some embodiments,the subject is not breastfeeding. In some embodiments, the subject isnot being treated with an anti-inflammatory drug. In some embodiments,the subject does not have an active infection (e.g., sepsis, pneumonia,abscess). In some embodiments, the subject does not have renal or liverimpairment (e.g., for women a serum creatinine level ≥125 μmol/L, formen a serum creatinine level of ≥125 μmol/L, an alanine aminotransferase(ALT) and aspartate aminotransferase (AST) ≥1.5× or 2× the upper limitof normal (ULN), alkaline phosphatase (ALP) and/or bilirubin >1.5×ULN.

In certain embodiments, the bacterial composition suppresses the immuneresponse in delayed-type hypersensitivity (DTH). In certain embodiments,the bacterial composition induces a regulatory T cell or ananti-inflammatory response. In certain embodiments, the bacterialcomposition inhibits antigen-specific responses. In certain embodiments,the bacterial composition treats allergic contact dermatitis. In certainembodiments, the bacterial composition treats autoimmune myocarditis. Incertain embodiments, the bacterial composition treats diabetes mellitustype 1. In certain embodiments, the bacterial composition treatsgranulomas. In certain embodiments, the bacterial composition treatsperipheral neuropathies. In certain embodiments, the bacterialcomposition treats Hashimoto's thyroiditis. In certain embodiments, thebacterial composition treats multiple sclerosis. In certain embodiments,the bacterial composition treats rheumatoid arthritis.

In certain embodiments, the bacterial composition treats inflammation ofthe colon. In certain embodiments, the bacterial composition treatscolitis. Colitis may be acute and self-limited or long-term. In certainembodiments, the bacterial composition treats ulcerative colitis. Incertain embodiments, the bacterial composition treats digestivediseases. In certain embodiments, the bacterial composition treatsCrohn's disease. In certain embodiments, the bacterial compositiontreats inflammatory bowel disease (IBD). In certain embodiments, thebacterial composition treats microscopic colitis. In certainembodiments, the bacterial composition treats collagenous colitis. Incertain embodiments, the bacterial composition treats diversion colitis.In certain embodiments, the bacterial composition treats chemicalcolitis. In certain embodiments, the bacterial composition treatsischemic colitis. In certain embodiments, the bacterial compositiontreats indeterminate colitis. In certain embodiments, the bacterialcomposition treats atypical colitis. In some embodiments, the methodfurther comprises administering to the subject an additional therapeutic(e.g., an antibiotic an immune suppressant, an anti-inflammatory agent).In some embodiments, the method further comprises administering to thesubject is a second therapeutic bacterium.

In some embodiments, the subject is a mammal. In some embodiments, thesubject is a human. In some embodiments, the subject is a non-humanmammal (e.g., a dog, a cat, a cow, a horse, a pig, a donkey, a goat, acamel, a mouse, a rat, a guinea pig, a sheep, a llama, a monkey, agorilla or a chimpanzee).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the efficacy of orally administered Lactococcus lactiscremoris Strain A in reducing antigen-specific ear swelling (earthickness) compared to vehicle (negative control), anti-inflammatoryDexamethasone (positive control), and Bacteria A, B, and C in a delayedtype hypersensitivity mouse model.

FIG. 2 is a line graphing showing percent weight change in acuteDSS-induced colitis model over a 12 day period for Lactococcus lactiscremoris Strain A in comparison to Bacteria A, B, and C, positivecontrol (anti-p40), and negative control (Sucrose vehicle). TheLactococcus lactis cremoris Strain A group showed less weight changethan the anti-p40 antibody (positive control).

FIG. 3A and FIG. 3B are plots showing that orally administeredLactococcus lactis cremoris Strain A reduces antigen-specific earswelling (ear thickness) compared to vehicle (negative control) andDexamethasone (FIG. 3A) and Fingolimod (FIG. 3B).

FIG. 4 is a plot showing the efficacy of Lactococcus lactis cremorisStrain A (with and without a 13 kb plasmid) and Lactococcus lactiscremoris Strain B (with and without a 30 kb plasmid) in reducingantigen-specific ear swelling (ear thickness) compared to vehicle andDexamethasone in a KLH-based delayed type hypersensitivity mouse model.Lactococcus lactis cremoris Strain A without a 13 kb plasmid has reducedefficacy compared Lactococcus lactis cremoris Strain A with a 13 kbplasmid. Conversely, removal of a 30 kb plasmid from L. lactis cremorisStrain B enhances efficacy compared to L. lactis cremoris Strain B withthe 30 kb plasmid.

FIG. 5 shows the efficacy of Lactococcus lactis cremoris Strain A inreducing antigen-specific ear swelling (ear thickness) compared tovehicle (negative control), and anti-inflammatory Dexamethasone(positive control) in an OVA based adoptive transfer delayed-typehypersensitivity (AdDTH) Mouse Model.

FIGS. 6A, 6B, and 6C show the ability of Lactococcus lactis cremorisStrain A in reducing expression of IL-12p70 (FIG. 6A), IL-22 (FIG. 6B),and KC (FIG. 6C) in an Adoptive Transfer Delayed-Type Hypersensitivity(AdDTH) Mouse Model. Circle represents vehicle, square representsdexamethasone, and triangle represents Lactococcus lactis cremorisStrain A.

FIG. 7 shows the efficacy of Lactococcus lactis cremoris Strain A inimproving the skin inflammation scores in an imiquimod model ofpsoriasis compared to control cream, vehicle, and dexamethasone.

FIG. 8 shows the efficacy of gamma-irradiated Lactococcus lactiscremoris Strain A in reducing antigen-specific ear swelling (earthickness) at 24 hours compared to vehicle (negative control) andanti-inflammatory Dexamethasone (positive control) in a KLH-baseddelayed type hypersensitivity mouse model. As shown, gamma-irradiatedLactococcus lactis cremoris Strain A retains efficacy.

FIGS. 9A, 9B, 9C, and 9D show the ability of gamma-irradiatedLactococcus lactis cremoris Strain A to reduce expression of IL-12p′70(FIG. 9A), TNF (FIG. 9B), IL-6 (FIG. 9C), and IL-13 (FIG. 9D) in aKLH-based delayed type hypersensitivity mouse model. Circle representsvehicle, square represents dexamethasone, and triangle representsgamma-irradiated Lactococcus lactis cremoris Strain A. Gamma-irradiatedLactococcus lactis cremoris Strain A decreases pro-inflammatory cytokineresponses in leukocytes from the site-draining lymph node. Circlerepresents vehicle, square represents dexamethasone, and trianglerepresents Lactococcus lactis cremoris Strain A.

FIGS. 10A and 10B show the ability of gamma-irradiated Lactococcuslactis cremoris Strain A to reduce the secretion of pro-inflammatorycytokines (IL-6 and TNFa) from gut-draining lymph nodes (FIG. 10A),while gamma-irradiated Lactococcus lactis cremoris Strain A inducesperipheral immune cells to secrete more IL-10 (FIG. 10B).

DETAILED DESCRIPTION General

In certain aspects, provided herein are methods and compositions (e.g.,bacterial compositions, pharmaceutical compositions) related to thetreatment and/or prevention of disease (e.g., cancer, autoimmunedisease, inflammatory disease, metabolic disease), in a subject (e.g., ahuman subject) comprising administering a bacterial compositioncomprising Lactococcus bacteria and/or a product of such bacteria (e.g.,extracellular vesicles (EVs) and/or pharmaceutically active biomasses(PhABs)). In certain aspects, also provided herein are methods oftreating an immune disorder (e.g., an autoimmune disease, aninflammatory disease, an allergy) in a subject comprising administeringto the subject a bacterial composition comprising an immune modulatingLactococcus strain provided herein, EVs generated by or isolated from animmune modulating Lactococcus strain provided herein and/or a PhAB madefrom or comprising an immune modulating Lactococcus strain providedherein.

Definitions

“Administration” broadly refers to a route of administration of acomposition to a subject. Examples of routes of administration includeoral administration, rectal administration, topical administration,inhalation (nasal) or injection. Administration by injection includesintravenous (IV), intramuscular (IM), intratumoral (IT) and subcutaneous(SC) administration. The pharmaceutical compositions described hereincan be administered in any form by any effective route, including butnot limited to intratumoral, oral, parenteral, enteral, intravenous,intraperitoneal, topical, transdermal (e.g., using any standard patch),intradermal, ophthalmic, (intra)nasally, local, non-oral, such asaerosol, inhalation, subcutaneous, intramuscular, buccal, sublingual,(trans)rectal, vaginal, intra-arterial, and intrathecal, transmucosal(e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal(e.g., trans- and perivaginally), intravesical, intrapulmonary,intraduodenal, intragastrical, and intrabronchial. In preferredembodiments, the pharmaceutical compositions described herein areadministered orally, rectally, intratumorally, topically,intravesically, by injection into or adjacent to a draining lymph node,intravenously, by inhalation or aerosol, or subcutaneously.

As used herein, the term “antibody” may refer to both an intact antibodyand an antigen binding fragment thereof. Intact antibodies areglycoproteins that include at least two heavy (H) chains and two light(L) chains inter-connected by disulfide bonds. Each heavy chain includesa heavy chain variable region (abbreviated herein as V_(H)) and a heavychain constant region. Each light chain includes a light chain variableregion (abbreviated herein as V_(L)) and a light chain constant region.The V_(H) and V_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each V_(H) and V_(L) is composed of three CDRs and fourFRs, arranged from amino-terminus to carboxy-terminus in the followingorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of theheavy and light chains contain a binding domain that interacts with anantigen. The term “antibody” includes, for example, monoclonalantibodies, polyclonal antibodies, chimeric antibodies, humanizedantibodies, human antibodies, multispecific antibodies (e.g., bispecificantibodies), single-chain antibodies and antigen-binding antibodyfragments.

The terms “antigen binding fragment” and “antigen-binding portion” of anantibody, as used herein, refers to one or more fragments of an antibodythat retain the ability to bind to an antigen. Examples of bindingfragments encompassed within the term “antigen-binding fragment” of anantibody include Fab, Fab′, F(ab′)2, Fv, scFv, disulfide linked Fv, Fd,diabodies, single-chain antibodies, NANOBODIES®, isolated CDRH3, andother antibody fragments that retain at least a portion of the variableregion of an intact antibody. These antibody fragments can be obtainedusing conventional recombinant and/or enzymatic techniques and can bescreened for antigen binding in the same manner as intact antibodies.

“Cellular augmentation” broadly refers to the influx of cells orexpansion of cells in an environment that are not substantially presentin the environment prior to administration of a composition and notpresent in the composition itself. Cells that augment the environmentinclude immune cells, stromal cells, bacterial and fungal cells.Environments of particular interest are the microenvironments wherecancer cells reside or locate. In some instances, the microenvironmentis a tumor microenvironment or a tumor draining lymph node. In otherinstances, the microenvironment is a pre-cancerous tissue site or thesite of local administration of a composition or a site where thecomposition will accumulate after remote administration.

“Clade” refers to the OTUs or members of a phylogenetic tree that aredownstream of a statistically valid node in a phylogenetic tree. Theclade comprises a set of terminal leaves in the phylogenetic tree thatis a distinct monophyletic evolutionary unit and that share some extentof sequence similarity. “Operational taxonomic units,” “OTU” (or plural,“OTUs”) refer to a terminal leaf in a phylogenetic tree and is definedby a nucleic acid sequence, e.g., the entire genome, or a specificgenetic sequence, and all sequences that share sequence identity to thisnucleic acid sequence at the level of species. In some embodiments thespecific genetic sequence may be the 16S sequence or a portion of the16S sequence. In other embodiments, the entire genomes of two entitiesare sequenced and compared. In another embodiment, select regions suchas multilocus sequence tags (MLST), specific genes, or sets of genes maybe genetically compared. In 16S embodiments, OTUs that share ≥97%average nucleotide identity across the entire 16S or some variableregion of the 16S are considered the same OTU (see e.g. Claesson M J,Wang Q, O'Sullivan O, Greene-Diniz R, Cole J R. Ros R P, and O'Toole PW. 2010. Comparison of two next-generation sequencing technologies forresolving highly complex microbiota composition using tandem variable16S rRNA gene regions. Nucleic Acids Res 38: e200. Konstantinidis K T,Ramette A, and Tiedje J M. 2006. The bacterial species definition in thegenomic era. Philos Trans R Soc Lond B Biol Sci 361: 1929-1940). Inembodiments involving the complete genome. MLSTs, specific genes, orsets of genes OTUs that share ≥95% average nucleotide identity areconsidered the same OTU (see e.g. Achtman M, and Wagner M. 2008.Microbial diversity and the genetic nature of microbial species. Nat.Rep. Microbiol. 6: 431-440. Konstantinidis K T, Ramette A, and Tiedje JM. 2006. The bacterial species definition in the genomic era. PhilosTrans R Sew Lond B Biol Sci 361: 1929-1940). OTUs are frequently definedby comparing sequences between organisms. Generally, sequences with lessthan 95% sequence identity are not considered to form part of the sameOTU. OTUs may also be characterized by any combination of nucleotidemarkers or genes, in particular highly conserved genes (e.g.,“house-keeping” genes), or a combination thereof. Such characterizationemploys, e.g., WGS data or a whole genome sequence.

A “combination” of two or more microbial strains includes the physicalco-existence of the two microbial strains, either in the same materialor product or in physically connected products, as well as the temporalco-administration or co-localization of the monoclonal microbialstrains.

The term “decrease” or “deplete” means a change, such that thedifference is, depending on circumstances, at least 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 1/100, 1/1000, 1/10,000, 1/100,000, 1/1,000,000or undetectable after treatment when compared to a pre-treatment state.

The term “ecological consortium” is a group of bacteria which tradesmetabolites and positively co-regulates one another, in contrast to twobacteria which induce host synergy through activating complementary hostpathways for improved efficacy.

The term “epitope” means a protein determinant capable of specificbinding to an antibody. Epitopes usually consist of chemically activesurface groupings of molecules such as amino acids or sugar side chains.Certain epitopes can be defined by a particular sequence of amino acidsto which an antibody is capable of binding.

As used herein, “engineered bacteria” are any bacteria that have beengenetically altered from their natural state by human intervention andthe progeny of any such bacteria. Engineered bacteria include, forexample, the products of targeted genetic modification, the products ofrandom mutagenesis screens and the products of directed evolution.

As used herein, the term “extracellular vesicle” or “EV” or refers to acomposition derived from a bacteria that comprises bacterial lipids, andbacterial proteins and/or bacterial nucleic acids and/or carbohydratemoieties contained in a nanoparticle. These EVs may contain 1, 2, 3, 4,5, 10, or more than 10 different lipid species. EVs may contain 1, 2, 3,4, 5, 10, or more than 10 different protein species. EVs may contain 1,2, 3, 4, 5, 10, or more than 10 different nucleic acid species. EVs maycontain 1, 2, 3, 4, 5, 10, or more than 10 different carbohydratespecies.

The term “gene” is used broadly to refer to any nucleic acid associatedwith a biological function. The term “gene” applies to a specificgenomic sequence, as well as to a cDNA or an mRNA encoded by thatgenomic sequence.

“Identity” as between nucleic acid sequences of two nucleic acidmolecules can be determined as a percentage of identity using knowncomputer algorithms such as the “FASTA” program, using for example, thedefault parameters as in Pearson et al. (1988) Proc. Natl. Acad. Sci.USA 85:2444 (other programs include the GCG program package (Devereux,J., et al., Nucleic Acids Research 12(I):387 (1984)), BLASTP, BLASTN,FASTA Atschul, S. F., et al., J Molec Biol 215:403 (1990); Guide to HugeComputers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, andCarillo et al. (1988) SIAM J Applied Math 48:1073). For example, theBLAST function of the National Center for Biotechnology Informationdatabase can be used to determine identity. Other commercially orpublicly available programs include, DNAStar “MegAlign” program(Madison, Wis.) and the University of Wisconsin Genetics Computer Group(UWG) “Gap” program (Madison Wis.)).

As used herein, the term “immune disorder” refers to any disease,disorder or disease symptom caused by an activity of the immune system,including autoimmune diseases, inflammatory diseases and allergies.Immune disorders include, but are not limited to, autoimmune diseases(e.g., Lupus, Scleroderma, hemolytic anemia, vasculitis, type onediabetes, Grave's disease, rheumatoid arthritis, multiple sclerosis,Goodpasture's syndrome, pernicious anemia and/or myopathy), inflammatorydiseases (e.g., acne vulgaris, asthma, celiac disease, chronicprostatitis, glomerulonephritis, inflammatory bowel disease, pelvicinflammatory disease, reperfusion injury, rheumatoid arthritis,sarcoidosis, transplant rejection, vasculitis and/or interstitialcystitis), and/or an allergies (e.g., food allergies, drug allergiesand/or environmental allergies).

The term “increase” means a change, such that the difference is,depending on circumstances, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 2-fold, 4-fold, 10-fold, 100-fold, 10{circumflex over ( )}3fold, 10{circumflex over ( )}4 fold, 10{circumflex over ( )}5 fold,10{circumflex over ( )}6 fold, and/or 10{circumflex over ( )}7 foldgreater after treatment when compared to a pre-treatment state.Properties that may be increased include immune cells, bacterial cells,stromal cells, myeloid derived suppressor cells, fibroblasts,metabolites, and cytokines.

The “internal transcribed spacer” or “ITS” is a piece of non-functionalRNA located between structural ribosomal RNAs (rRNA) on a commonprecursor transcript often used for identification of eukaryotic speciesin particular fungi. The rRNA of fungi that forms the core of theribosome is transcribed as a signal gene and consists of the 8S, 5.8Sand 28S regions with ITS4 and 5 between the 8S and 5.8S and 5.8S and 28Sregions, respectively. These two intercistronic segments between the 18Sand 5.8S and 5.8S and 28S regions are removed by splicing and containsignificant variation between species for barcoding purposes aspreviously described (Schoch et al Nuclear ribosomal internaltranscribed spacer (ITS) region as a universal DNA barcode marker forFungi. PNAS 109:6241-6246. 2012). 18S rDNA is traditionally used forphylogenetic reconstruction however the ITS can serve this function asit is generally highly conserved but contains hypervariable regions thatharbor sufficient nucleotide diversity to differentiate genera andspecies of most fungus.

The term “isolated” or “enriched” encompasses a microbe, bacteria orother entity or substance that has been (1) separated from at least someof the components with which it was associated when initially produced(whether in nature or in an experimental setting), and/or (2) produced,prepared, purified, and/or manufactured by the hand of man. Isolatedmicrobes may be separated from at least about 10%, about 20%, about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, ormore of the other components with which they were initially associated.In some embodiments, isolated microbes are more than about 80%, about85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%,about 96%, about 97%, about 98%, about 99%, or more than about 99% pure.As used herein, a substance is “pure” if it is substantially free ofother components. The terms “purify,” “purifying” and “purified” referto a microbe or other material that has been separated from at leastsome of the components with which it was associated either wheninitially produced or generated (e.g., whether in nature or in anexperimental setting), or during any time after its initial production.A microbe or a microbial population may be considered purified if it isisolated at or after production, such as from a material or environmentcontaining the microbe or microbial population, and a purified microbeor microbial population may contain other materials up to about 10%,about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about80%, about 90%, or above about 90% and still be considered “isolated.”In some embodiments, purified microbes or microbial population are morethan about 80%, about 85%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, ormore than about 99% pure. In the instance of microbial compositionsprovided herein, the one or more microbial types present in thecomposition can be independently purified from one or more othermicrobes produced and/or present in the material or environmentcontaining the microbial type. Microbial compositions and the microbialcomponents thereof are generally purified from residual habitatproducts.

“Metabolite” as used herein refers to any and all molecular compounds,compositions, molecules, ions, co-factors, catalysts or nutrients usedas substrates in any cellular or microbial metabolic reaction orresulting as product compounds, compositions, molecules, ions,co-factors, catalysts or nutrients from any cellular or microbialmetabolic reaction.

“Microbe” refers to any natural or engineered organism characterized asa bacterium, fungus, microscopic alga, protozoan, and the stages ofdevelopment or life cycle stages (e.g., vegetative, spore (includingsporulation, dormancy, and germination), latent, biofilm) associatedwith the organism. Examples of gut microbes include: Actinomycesgraevenitzii, Actinomyces odontolyticus, Akkermansia muciniphila,Bacteroides caccae, Bacteroides fragilis, Bacteroides putredinis,Bacteroides thetaiotaomicron, Bacteroides vultagus, Bifidobacteriumadolescentis, Bifidobacterium bifidum, Bilophila wadsworthia,Lactococcus lactis, Butyrivibrio, Campylobacter gracilis, Clostridiacluster III, Clostridia cluster IV, Clostridia cluster IX(Acidaminococcaceae group), Clostridia cluster XI, Clostridia clusterXIII (Peptostreptococcus group), Clostridia cluster XIV, Clostridiacluster XV, Collinsella aerofaciens, Coprococcus, Corynebacteriumsunsvallense, Desulfomonas pigra, Dorea formicigenerans, Dorealongicatena, Escherichia coli, Eubacterium hadrum, Eubacterium rectale,Faecalibacteria prausnitzii, Gemella, Lactococcus, Lanchnospira,Mollicutes cluster XVI, Mollicutes cluster XVIII, Prevotella, Rothiamucilaginosa, Ruminococcus callidus, Ruminococcus gnavus, Ruminococcustorques, and Streptococcus.

“Microbiome” broadly refers to the microbes residing on or in body siteof a subject or patient. Microbes in a microbiome may include bacteria,viruses, eukaryotic microorganisms, and/or viruses. Individual microbesin a microbiome may be metabolically active, dormant, latent, or existas spores, may exist planktonically or in biofilms, or may be present inthe microbiome in sustainable or transient manner. The microbiome may bea commensal or healthy-state microbiome or a disease-state microbiome.The microbiome may be native to the subject or patient, or components ofthe microbiome may be modulated, introduced, or depleted due to changesin health state (e.g., precancerous or cancerous state) or treatmentconditions (e.g., antibiotic treatment, exposure to different microbes).In some aspects, the microbiome occurs at a mucosal surface. In someaspects, the microbiome is a gut microbiome. In some aspects, themicrobiome is a tumor microbiome.

A “microbiome profile” or a “microbiome signature” of a tissue or samplerefers to an at least partial characterization of the bacterial makeupof a microbiome. In some embodiments, a microbiome profile indicateswhether at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more bacterial strainsare present or absent in a microbiome.

“Modified” in reference to a bacteria broadly refers to a bacteria thathas undergone a change from its wild-type form. Examples of bacterialmodifications include genetic modification, gene expression, phenotypemodification, formulation, chemical modification, and dose orconcentration. Examples of improved properties are described throughoutthis specification and include, e.g., attenuation, auxotrophy, homing,or antigenicity. Phenotype modification might include, by way ofexample, bacteria growth in media that modify the phenotype of abacterium that increase or decrease virulence.

As used herein, a gene is “overexpressed” in a bacteria if it isexpressed at a higher level in an engineered bacteria under at leastsome conditions than it is expressed by a wild-type bacteria of the samespecies under the same conditions. Similarly, a gene is “underexpressed”in a bacteria if it is expressed at a lower level in an engineeredbacteria under at least some conditions than it is expressed by awild-type bacteria of the same species under the same conditions.

The terms “polynucleotide” and “nucleic acid” are used interchangeably.They refer to a polymeric form of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides, or analogs thereof.Polynucleotides may have any three-dimensional structure, and mayperform any function. The following are non-limiting examples ofpolynucleotides: coding or non-coding regions of a gene or genefragment, loci (locus) defined from linkage analysis, exons, introns,messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers. A polynucleotide may comprise modifiednucleotides, such as methylated nucleotides and nucleotide analogs. Ifpresent, modifications to the nucleotide structure may be impartedbefore or after assembly of the polymer. A polynucleotide may be furthermodified, such as by conjugation with a labeling component. In allnucleic acid sequences provided herein, U nucleotides areinterchangeable with T nucleotides.

As used herein, a substance is “pure” if it is substantially free ofother components. The terms “purify,” “purifying” and “purified” referto a EV or other material that has been separated from at least some ofthe components with which it was associated either when initiallyproduced or generated (e.g., whether in nature or in an experimentalsetting), or during any time after its initial production. An EV may beconsidered purified if it is isolated at or after production, such asfrom one or more other bacterial components, and a purified microbe ormicrobial population may contain other materials up to about 10%, about20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%,about 90%, or above about 90% and still be considered “purified.” Insome embodiments, purified EVs are more than about 80%, about 85%, about90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, about 99%, or more than about 99% pure. EVcompositions and the microbial components thereof are, e.g., purifiedfrom residual habitat products.

As used herein, the term “purified EV composition” or “EV composition”refer to a preparation that includes EVs that have been separated fromat least one associated substance found in a source material (e.g.separated from at least one other bacterial component) or any materialassociated with the EVs in any process used to produce the preparation.It also refers to a composition that has been significantly enriched orconcentrated. In some embodiments the EVs are concentrated by 2 fold,3-fold, 4-fold, 5-fold, 10-fold, 100-fold, 1000-fold, 10,000-fold ormore than 10,000 fold.

“Operational taxonomic units” and “OTU(s)” refer to a terminal leaf in aphylogenetic tree and is defined by a nucleic acid sequence, e.g., theentire genome, or a specific genetic sequence, and all sequences thatshare sequence identity to this nucleic acid sequence at the level ofspecies. In some embodiments the specific genetic sequence may be the16S sequence or a portion of the 16S sequence. In other embodiments, theentire genomes of two entities are sequenced and compared. In anotherembodiment, select regions such as multilocus sequence tags (MLST),specific genes, or sets of genes may be genetically compared. For 16S,OTUs that share ≥97% average nucleotide identity across the entire 16Sor some variable region of the 16S are considered the same OTU. See e.g.Claesson M J, Wang Q, O'Sullivan O, Greene-Diniz R, Cole J R, Ross R P,and O'Toole P W. 2010. Comparison of two next-generation sequencingtechnologies for resolving highly complex microbiota composition usingtandem variable 16S rRNA gene regions. Nucleic Acids Res 38: e200.Konstantinidis K T, Ramette A, and Tiedje J M. 2006. The bacterialspecies definition in the genomic era. Philos Trans R Soc Lond B BiolSci 361: 1929-1940. For complete genomes, MLSTs, specific genes, otherthan 16S, or sets of genes OTUs that share ≥95% average nucleotideidentity are considered the same OTU. See e.g., Achtman M, and Wagner M.2008. Microbial diversity and the genetic nature of microbial species.Nat. Rev. Microbiol. 6: 431-440. Konstantinidis K T, Ramette A, andTiedje J M. 2006. The bacterial species definition in the genomic era.Philos Trans R Soc Lond B Biol Sci 361: 1929-1940. OTUs are frequentlydefined by comparing sequences between organisms. Generally, sequenceswith less than 95% sequence identity are not considered to form part ofthe same OTU. OTUs may also be characterized by any combination ofnucleotide markers or genes, in particular highly conserved genes (e.g.,“house-keeping” genes), or a combination thereof. Operational TaxonomicUnits (OTUs) with taxonomic assignments made to, e.g., genus, species,and phylogenetic clade are provided herein.

As used herein, “specific binding” refers to the ability of an antibodyto bind to a predetermined antigen or the ability of a polypeptide tobind to its predetermined binding partner. Typically, an antibody orpolypeptide specifically binds to its predetermined antigen or bindingpartner with an affinity corresponding to a K_(D) of about 10⁻⁷ M orless, and binds to the predetermined antigen/binding partner with anaffinity (as expressed by K_(D)) that is at least 10 fold less, at least100 fold less or at least 1000 fold less than its affinity for bindingto a non-specific and unrelated antigen/binding partner (e.g., BSA,casein). Alternatively, specific binding applies more broadly to a twocomponent system where one component is a protein, lipid, orcarbohydrate or combination thereof and engages with the secondcomponent which is a protein, lipid, carbohydrate or combination thereofin a specific way.

The terms “subject” or “patient” refers to any animal. A subject or apatient described as “in need thereof” refers to one in need of atreatment for a disease. Mammals (i.e., mammalian animals) includehumans, laboratory animals (e.g., primates, rats, mice), livestock(e.g., cows, sheep, goats, pigs), and household pets (e.g., dogs, cats,rodents). For example, the subject may be a non-human mammal includingbut not limited to of a dog, a cat, a cow, a horse, a pig, a donkey, agoat, a camel, a mouse, a rat, a guinea pig, a sheep, a llama, a monkey,a gorilla or a chimpanzee. The subject or patient may be healthy, or maybe suffering from an immune disorder at any developmental stage.

“Strain” refers to a member of a bacterial species with a geneticsignature such that it may be differentiated from closely-relatedmembers of the same bacterial species. The genetic signature may be theabsence of all or part of at least one gene, the absence of all or partof at least on regulatory region (e.g., a promoter, a terminator, ariboswitch, a ribosome binding site), the absence (“curing”) of at leastone native plasmid, the presence of at least one recombinant gene, thepresence of at least one mutated gene, the presence of at least oneforeign gene (a gene derived from another species), the presence atleast one mutated regulatory region (e.g., a promoter, a terminator, ariboswitch, a ribosome binding site), the presence of at least onenon-native plasmid, the presence of at least one antibiotic resistancecassette, or a combination thereof. Genetic signatures between differentstrains may be identified by PCR amplification optionally followed byDNA sequencing of the genomic region(s) of interest or of the wholegenome. In the case in which one strain (compared with another of thesame species) has gained or lost antibiotic resistance or gained or losta biosynthetic capability (such as an auxotrophic strain), strains maybe differentiated by selection or counter-selection using an antibioticor nutrient/metabolite, respectively.

As used herein, the term “treating” a disease in a subject or “treating”a subject having or suspected of having a disease refers to subjectingthe subject to a pharmaceutical treatment, e.g., the administration ofone or more agents, such that at least one symptom of the disease isdecreased or prevented from worsening. Thus, in one embodiment,“treating” refers inter alia to delaying progression, expeditingremission, inducing remission, augmenting remission, speeding recovery,increasing efficacy of or decreasing resistance to alternativetherapeutics, or a combination thereof.

Bacteria

In certain aspects, provided herein are methods of using a bacterialcomposition comprising an immune modulating Lactococcus strain providedherein, EVs generated by or isolated from an immune modulatingLactococcus strain provided herein and/or a PhAB made from or comprisingan immune modulating Lactococcus strain provided herein. In someembodiments, the immune modulating Lactococcus strain is a strain ofLactococcus lactis cremoris. In some embodiments, the immune modulatingLactococcus strain is Lactococcus lactis cremoris Strain A (ATCC DepositNumber PTA-125368). In some embodiments, the immune modulatingLactococcus strain is a strain comprising at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% sequence identity (e.g., atleast 99.5% sequence identity, at least 99.6% sequence identity, atleast 99.7% sequence identity, at least 99.8% sequence identity, atleast 99.9% sequence identity) to the nucleotide sequence (e.g.,genomic, 16S or CRISPR nucleotide sequence) of the Lactococcus lactiscremoris Strain A (ATCC Deposit Number PTA-125368).

Under the terms of the Budapest Treaty on the International Recognitionof the Deposit of Microorganisms for the Purpose of Patent Procedure,the Lactococcus lactis cremoris Strain A was deposited on Oct. 11, 2018,with the American Type Culture Collection (ATCC) of 10801 UniversityBoulevard, Manassas, Va. 20110-2209 USA and was assigned ATCC AccessionNumber PTA-125368.

Applicant represents that the ATCC is a depository affording permanenceof the deposit and ready accessibility thereto by the public if a patentis granted. All restrictions on the availability to the public of thematerial so deposited will be irrevocably removed upon the granting of apatent. The material will be available during the pendency of the patentapplication to one determined by the Commissioner to be entitled theretounder 37 CFR 1.14 and 35 U.S.C. 122. The deposited material will bemaintained with all the care necessary to keep it viable anduncontaminated for a period of at least five years after the most recentrequest for the furnishing of a sample of the deposited plasmid, and inany case, for a period of at least thirty (30) years after the date ofdeposit or for the enforceable life of the patent, whichever period islonger. Applicant acknowledges its duty to replace the deposit shouldthe depository be unable to furnish a sample when requested due to thecondition of the deposit.

In some embodiments, the bacteria described herein are modified toimprove colonization and/or engraftment in the mammaliangastrointestinal tract (e.g., modified metabolism, such as improvedmucin degradation, enhanced competition profile, increased motility,increased adhesion to gut epithelial cells, modified chemotaxis). Insome embodiments, the bacteria described herein are modified to enhancetheir immunomodulatory and/or therapeutic effect (e.g., either alone orin combination with another therapeutic agent). In some embodiments, thebacteria described herein are modified to enhance immune activation(e.g., through modified production of polysaccharides, pili, fimbriae,adhesins). In some embodiments, the bacteria described herein aremodified to improve bacterial manufacturing (e.g., higher oxygentolerance, improved freeze-thaw tolerance, shorter generation times).

Lactococcus lactis cremoris Strain A can be cultured according tomethods known in the art. For example, Lactococcus lactis cremoris canbe grown in ATCC Medium 2722, ATCC Medium 1490, or other medium usingmethods disclosed, for example in Caballero et al., 2017. “CooperatingCommensals Restore Colonization Resistance to Vancomycin-ResistantEnterococcus faecium” Cell Host & Microbe 21:592-602, which is herebyincorporated by reference in its entirety.

In some embodiments, the immune modulating Lactococcus bacteria is astrain of Lactococcus bacteria (e.g., a strain of Lactococcus lactiscremoris) comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more)proteins listed in Table 1 and/or one or more (e.g., 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25or more) genes encoding proteins listed in Table 1. In some embodiments,the immune modulating bacteria comprises all of the proteins listed inTable 1 and/or all of the genes encoding the proteins listed in Table 1.

In some embodiments, the immune modulating Lactococcus bacteria is astrain of Lactococcus bacteria (e.g., a strain of Lactococcus lactiscremoris) comprising one or more (e.g., one, two or three) membraneassociated proteins listed in Table 2 and/or one or more (e.g., one, twoor three) genes encoding membrane associated proteins listed in Table 2.In some embodiments, the immune modulating bacteria comprises all of theproteins listed in Table 2 and/or all of the genes encoding the proteinslisted in Table 2.

In some embodiments, the immune modulating Lactococcus bacteria is astrain of Lactococcus bacteria (e.g., a strain of Lactococcus lactiscremoris) free or substantially free of one or more (e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25 or more) proteins listed in Table 3 and/or one or more (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25 or more) genes encoding proteins listed in Table 3. Insome embodiments, the immune modulating bacteria is free of all of theproteins listed in Table 2 and/or all of the genes encoding the proteinslisted in Table 2.

In some embodiments, the immune modulating Lactococcus bacteria is astrain of Lactococcus bacteria (e.g., a strain of Lactococcus lactiscremoris) free or substantially free of one or more (e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17) exopolysaccharide (EPS)synthesis proteins listed in Table 4 and/or one or more (e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17) genes encoding EPSsynthesis proteins listed in Table 4. In some embodiments, the immunemodulating bacteria is free of all of the proteins listed in Table 4and/or all of the genes encoding the proteins listed in Table 4. In someembodiments, the immune modulating bacteria is a strain of Lactococcusbacteria (e.g., a strain of Lactococcus lactis cremoris) free orsubstantially free of an EPS synthesized in whole or in part by aprotein listed in Table 4. In some embodiments, the immune modulatingbacteria is a strain of Lactococcus bacteria (e.g., a strain ofLactococcus lactis cremoris) free or substantially free of EPS.

In certain aspects, the immune modulating Lactococcus strain bacteriadescribed herein are substantially free of exopolysaccharides.

In some embodiments, the immune modulating Lactococcus bacteria is astrain of Lactococcus bacteria (e.g., a strain of Lactococcus lactiscremoris) comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more)proteins listed in Table 6 and/or one or more (e.g., 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25or more) genes encoding proteins listed in Table 6. In some embodiments,the immune modulating bacteria comprises all of the proteins listed inTable 6 and/or all of the genes encoding the proteins listed in Table 6.

In some embodiments, the immune modulating Lactococcus bacteria is astrain of Lactococcus bacteria (e.g., a strain of Lactococcus lactiscremoris) free or substantially free of one or more (e.g., 1, 2, 3, 4,5, 6, 7, or more) proteins listed in Table 5 and/or one or more (e.g.,1, 2, 3, 4, 5, 6, 7 or more) genes encoding proteins listed in Table 5.In some embodiments, the immune modulating bacteria is free of all ofthe proteins listed in Table 5 and/or all of the genes encoding theproteins listed in Table 5.

TABLE 1 Exemplary Lactococcus lactis cremoris Strain A Proteins SEQ IDNO. name uniprot_id Protein Sequence 1 Cluster: P35881MTQFTTELLNFLAQKQDIDEFFRTSLETAMNDLLQAE TransposaseLSAFLGYEPYDKVGYNSGNSRNGSYSRQFETKYGT for insertionVQLSIPRDRNGNFSPALLPAYGRRDDHLEEMVIKLY sequenceQTGVTTREISDIIERMYGHHYSPATISNISKATQENVA elementTFHERSLEANYSVLFLDGTYLPLRRGTVSKECIHIAL IS905GITPEGQKAVLGYEIAPNENNASWSTLLDKLQNQGIQQVSLVVTDGFKGLEQIISQAYPLAKQQRCLIHISRNLASKVKRADRAVILEQFKTIYRAENLEMAVQALENFIAEWKPKYRKVMESLENTDNLLTFYQFPYQIWHSIYSTNLIESLNKEIKRQTKKKVLFPNEEALERYLVTLFEDYN FKQSQRIHKGFGQCADTLESLFD 2Cluster: Q9CB06 MKVTGFPKATYYYWVNCFERVNKDELIEKEMLKIRQ TransposaseEHANAGYRPMSELLKQRGYHVNHKKVQRLMKKLGL of IS1077ERVTSYWHKSRKYNSYKGKVGTVAKNKLHRRFRTSIPHQKITTDTTEFKYYEDGIQKKCYLNPYIDLFNSEVISYHISKQPSYQSIDIALNQALAVTSDCPYRRTFHSDQG WGYQMRDYVSKLKSHRIFQSMSRKGNCHDNSVMENFFGLLKQEIYYGHIFSSFEELEQVIVIWIRYYNTKRIK QKLNWMSPIQFRLNYQNN 3 Cluster:T0VLJ3 MTQFTTELLNFLAQKQDIDEFFRTSLETAMNDLLQAE TransposaseLSAFLGYEPYDKVGYNSGNSRNGSYSRQFETKYGT IS256VQLSIPRDRNGNFSPALLPAYGRRDDHLEEMGYQTL SNRCNDSRNL 4 Cluster: A2RKL1MTKYSFELKLKVVQDYDNGVGGCDYLAKKYHVTNE UncharacterizedAIVRRWVKAYKELGAVGIQRKRQNTVYSTQFKLNAV proteinNLYLTSEKSYRELAHELGMNNPPLLTRWVSNYRKKGEFAFSNVQGRPRKESELLEISIKKAKDVVNETEQELA  RLQNDNLNLRMEVEYLKGLRRLRQEQHKRENPEWS VNSDENSSSHLSNS 5 Cluster: S6EVX2MVCELRREFKFPLKQLLAISELSKATYYYWVNRFER TransposasePNKDEMIEQVMLEIRQEHTNAGYRPMVELLKQRGIY VNHKKVQRLMKKLGLRVTTFWHKSRKYNSYKGKVGTVAKNKLHRRFNTSIPHQKITTDTTEFKYYDKGVQKKLYLTPYLDLFNNEVISYEISKQPTYQAIATALQEALELTSDCLYRRTFHSDQGWAYQMKNYVFKLKSQKIIQSM SRKGNCHDNSVMENFFGLLKQEIYYGHVFNSFEELEQAITKWIHYYNTKRIKKKLNWMSPIQYRLTYSK 6 Cluster: G0WJR5MMINYQGEVFTETEFYGREILEAIQLTNKFPTPKKVLI PIL4_5DRLEEMIHEQLDLIDKEELNNYIHAKK 7 Cluster: T0W7Q8MKIIENRERSIQKKFFVNEKENERIKLMMKKTGITNFS Molybdopterin-VFARRACCNKEIFTLDFSEYKNIISEISATKSELKRIGN guanineNINQIAKHLNENKNNQTESLMSDYQNQLESLEEKIQK dinucleotide VVHYISEG biosynthesisprotein MobC 8 ESAT-6 Q2G188 MMLKKEWQAILKHKFFIIVIIALALVPAIYNYIFLGSMWsecretion_ DPSGKLNDLPVAVVNLDKTSELNGKKFKLGDDVITE   accessory_MKKSKDLDYHFVSKDKASEGIKKGDYYMVITFPENF factor_EsaASENATTLMNKEPKTVQLDYQTTRGHNYISSKMSESA MNQLKSEVSKNITQTYTKTRIAS 9Foldase_protein_ P0C2B5 MKKKMRLKVLLASTATALLLLSGCQSNQTDQTVATY PrsASGGKVTESSFYKELKQSPTTKTMLANMLIYRALNHA YGKSVSTKTVNDAYDSYKQQYGENFDAFLSQNGFSRSSFKESLRTNFLSEVALKKLKKVSESQLKAAWKTYQPKVTVQHILTSDEDTAKQVISDLAAGKDFAMLAKTDSIDTATKDNGGKISFELNNKTLDATFKDAAYKLKNGDYTQTPVKVTDGYEVIKMINHPAKGTFTSSKKVLTASVYAKWSRDSSIMQRVISQVLKNQHVTIKDKDLADALD SYKKLATTN 10 PIII- P15292MQRKKKGLSFLLAGTVALGALAVLPVGEIQAKAAISQ type_proteinaseQTKGSSLANTVTAATAKQAATDTTAATTNQAIATQLAAKGIDYNKLNKVQQQDIYVDVIVQMSAAPASENGTLRTDYSSTAEIQQETNKVIAAQASVKAAVEQVTQQTAGESYGYVVNGFSTKVRVVDIPKLKQIAGVKTVTLAKVYYPTDAKANSMANVQAVWSNYKYKGEGTVVSVIDS GIDPTHKDMRLSDDKDVKLTKSDVEKFTDTVKHGRYFNSKVPYGFNYADNNDTITDDKVDEQHGMHVAGIIGANGTGDDPAKSVVGVAPEAQLLAMKVFTNSDTSATTGSDTLVSAIEDSAKIGADVLNMSLGSDSGNQTLEDPEIAAVQNANESGTAAVISAGNSGTSGSATEGVNKDYYGLQDNEMVGTPGTSRGATTVASAENTDVITQAVTITDGTGLQLGPETIQLSSNDFTGSFDQKKFYVVKDASGNLSKGKVADYTADAKGKIAIVKRGELTFDDKQKYAQAAGAAGLIIVNNDGTATPVTSMALTTTFPTFGLSSVTGQKLVDWVTAHPDDSLGVKIALTLVPNQKYTEDKMSDFTSYGPVSNLSFKPDITAPGGNIWSTQNNNGYTNMSGTSMASPFIAGSQALLKQALNNKNNPFYAYYKQLKGTALTDFLKTVEMNTAQPINDINYNNVIVSPRRQGAGLVDVKAAIDALEKNPSTVVAENGYPAVELKDFTSTDKTFKLTFTNRTTHELTYQMDSNTDTNAVYTSATDPNSGVLYDKKIDGAAIKAGSNITVPAGKTAQIEFTLSLPKSFDQQQFVEGFLNFKGSDGSRLNLPYMGFFGDWN DGKIVDSLNGITYSPAGGNFGTVPLLTNKNTGTQYYGGMVIDADGNQTVDDQA1AFSSDKNALYNDISMKYYLLRNISNVQVDILDGQGNKVTTLSSSTNLTKTYYNAHSQQYIYYHAPAWDGTYYDQRDGNIKTADDGSYTYRISGVPEGGDKRQVFDVPFKLDSKAPTVRHVALSAKTKNGKTQYYLTAEVKDDLSGLDATKSVKTAINEVTNLDATFTDAGTTADGYTKIETPLSDEQAQALGNGDNSAELYLTDNASNATDQDASVQKPGSTSFDLIVNGSGIPDKISSTTTGYEANTQGGGTYTFSGTYPAAVDGTYTDAQ GKKHDLNTTYDAATNSFTASMPVTNADYAAQVDLYADKAHTQLLKHFDTKVRLTAPTFTDLKFNNGSDQTSEATIKVTGTVSADTKTVNVGDTVAALDAQHHFSVDVPVNYGDNTIKVIATDEDGNTTTEQKTITSSYDPDMLKNPVTFDQGVTFGSNEFNATSAKFYDPKTGIATITGKVKHPTTTLQVDGKQIPIKDDLTFSFTLDLGTLGQKPFGVVVGDTTQNKTFQEALTFILDAVAPTLSLDSSTDAPVYTNDPNFQITGTATDNAQYLSLSINGSSVASQYADININSGKPGHMAIDQPVKLLEGKNVLTVAVTDSEDNTTTKNITVYYEPKKTLAAPTVTPSTTEPAQTVTLTANAAATGETVQYSADGGKTYQDVPAAGVTITANGTFKFKSTDLYGNESPAVDYVVTNIKADDPAQLQAAKQALTNLIASAKTLSASGKYDDATTTALAAATQKAQTALDQTNASVDSLTGANRDLQTAINQLAAKLPADKKTSLLNQLQSVKDALGTDLGNQTDPSTGKTFTAALDDLVAQAQAGTQTDDQLQATLAKILDEVLAKLAEGIKAATPAEVGNAKDAATGKTWYADIADTLTSGQASADASDKLAHLQALQSL KTKVAAAVEADKTVGKGDDTTGTSDKGSGQGTPAPATGDTGKDKGDEGSQPSSGGNIPTNPATTTSTSAD DTTDRNGQHTTGTSDKGGGQGTPAPATGDTGKDKGDEGSQPSSGGNIPTNPATTTSTSADDTTDRNGQH TTGTSDKGGGQGTPAPATGDTGKDKGDEGSQPSSGGNIPTNPATTTSTSTDDTTDRNGQHTTGKGALPKT GETTERPAFGFLGVIVVILMGVLGLKRKQREE 11Cluster: T0V9Y4 MRAAEGLFVYNKTNFHYLPQNIAFADFKSGKFATSG UncharacterizedMSMILIDSVNHRILDVMKDRGAGQLRAYFNQYSPSA proteinRAAVKTITVDLFTPYRAMIKDLFPNANIVADRFHVVTQAYRELNKVRISVMKQFGSDSKEYRQLKRFWKLLMKHENALDYMTSKNRINFKHAYLTDKEVIDRLLALSDEL RDAYAFYQVIL 12 Cluster: T0UTW8MDNDIRILIGLTDLNIDFDAKAEQHFNETNLNGTAPIT UncharacterizedWNLLLTYATNCEKFGTPMVHNGIKMVTHKGPRIAFK protein FQNYRIRKQKFL 13 Cluster:T0UZT2 MIENTINIAYARKFYKTKDYHSFCNLIKGNKGLFGNKT UncharacterizedVNQKANISFVKSEGEKHTHIYLDYQETCKVAHPNFLQ proteinLINLLKNYDPEFSEEKLPTFDLNDKIFGEYEIKVIPISKTKIVNTIDDVMNEIAKEIVLKYNQDMFKVTSKLGEISLT PIQEKFDKLKDI 14 Cluster: Q9AIQ4MIIPEKQNKQKQVLTLNELEKRKVVEHNALIQSVAKM RepBQKTALKMFELAVSCIDTEEPPKNNTVYLSKSELFKFFEVSSSSKHSQFKEAVNYMQKQAFFNIKADKKLGIEYESIVPIPYVKWNDYNDEVTIRFDQAIMPYLIDLKAEFTQYKISELQKLNSKYSIILYRWLSMNYNQYEHYSVKGGRRADQVEAYRTPSIKVKELREITDTINEHQHFPHFETRVLKKAIEEINAHTSFNVTYEKVKKGRSIDSIVFHIEKKRMADDNSYKLEDKVYQEDKARKAETEKDLVFQAMQSPYTRLLIENMFLNVYETTDSQIMAGLQKNVYPLYDELKELRGLNGVKDHLSYVSSKQEAYSKRNVAKYLKKA IEQYLPTVKRQDLNHE 15 Cluster: Q7BLH6MSEDLKTIKELADELGVSKSYVDKIIRILKLHTKLDKV UncharacterizedGNKYVISKKQEKSIITRIENSKSTTETHTESTTQSHTK proteinVDAEVDFLKEEIAYLKSNHDKQLTNKDKQIETLSNLLDQQQRLALQDKKWLEEYKAEINDLKALKMPSEDTKE  EQSNYRSLEKEKDFVQTIQESYESEIKVLNQKLAEQE EQIQEIQKEKETKEKKWFQFWK 16 Cluster:Q05547 MAQTFDRKILRALQDNGVREIRAYEVVSKRLTIFQTD RepCRGTFKYSDSLYRLVAPRQELWRNCTTGFISEEKYHF YKK 17 HypotheticalMNHFKGKQFKKDVIIVAVGYYLRYNLSYREIQELLYD proteinRGINVCHTTIYRWVQEYSKVLYHLWKKKNRQSFYSWKMDETYIKIKGRWHYLYRAIDADGLTLDIWLRKKRDTQAAYAFLKRLHKQFGQPRVIVTDKAPSIGSAFRKLQSNGLYTKTEHRTVKYLNNLIEQDHRPIKRRNKFYRSLRTASTTIKGMETIRGIYKKNRRNGTLFGFSVSTEIKVL MGILA 18 Cluster: Q52233MKEYFQGDEFKDISKNGKDRKWKERKINNLNLAKIF ReplicationDSLDYPDSFIFNIKSCAEYLNFKRSSDGSLRLFQMYT proteinCKNKQCAICSWRRSMKYQVQISKIVEEAMIRKPKGRFLFLTLTVENVSGEGLNNELSLLSEAFNRLMKYKKVSKNILGFLRATEVTINESMDTYHPHIHVLLFISPTYFKNKNNYISQDEWTELWKKSAKLDYRPIVDVRSIKPKNEKTSDIRSAILETAKYPVKPMELNYDSAKVVDDLQKGLYRKRQIAFGGLFKQIKKELELDDIENGDLINIGDEENPISD GEIISVLWNHERQNYYVR 19 Cluster:T0VLA4 MINYQGEDFTETEFYGREILEAIQLTNKFPTPKKVLID UncharacterizedMLEEMIHEQLDFIDKEELNNYINAKKYVQTLTEDEVK protein NLCFEVKDLYEDVLKEFEIKL 20Cluster: T0VQK1 MTCSNLTIHLHAKNRSKLFGSKKYALQELEAESTAFV UncharacterizedVANHLNIDTKDYSIGYLNSWGFDKISDEQLENVIKND protein KLSNNKIKGENE (Fragment)

TABLE 2 Selected membrane associated Lactococcus lactis cremoris StrainA Proteins SEQ ID NO. name uniprot_id Protein Sequence 21 ESAT-6_ Q2G188MMLKKEWQAILKHKFFIIVIIALALVPAIYNYIFLGSMW secretion_DPSGKLNDLPVAVVNLDKTSELNGKKFKLGDDVITE accessory_MKKSKDLDYHFVSKDKASEGIKKGDYYMVITFPENF factor_EsaASENATTLMNKEPKTVQLDYQTTRGHNYISSKMSESA MNQLKSEVSKNITQTYTKTRIAS 22 Foldase_P0C2B5 MKKKMRLKVLLASTATALLLLSGCQSNQTDQTVATY protein_PrsASGGKVTESSFYKELKQSPTTKTMLANMLIYRALNHA YGKSVSTKTVNDAYDSYKQQYGENFDAFLSQNGFSRSSFKESLRTNFLSEVALKKLKKVSESQLKAAWKTYQPKVTVQHILTSDEDTAKQVISDLAAGKDFAMLAKTDSIDTATKDNGGKISFELNNKTLDATFKDAAYKLKNGDYTQTPVKVTDGYEVIKMINHPAKGTFTSSKKVLTASVYAKWSRDSSIMQRVISQVLKNQHVTIKDKDLADALD SYKKLATTN 23 PIII- P15292MQRKKKGLSFLLAGTVALGALAVLPVGEIQAKAAISQ type_QTKGSSLANTVTAATAKQAATDTTAATTNQAIATQLA proteinaseAKGIDYNKLNKVQQQDIYVDVIVQMSAAPASENGTLRTDYSSTAEIQQETNKVIAAQASVKAAVEQVTQQTAGESYGYVVNGFSTKVRVVDIPKLKQIAGVKTVTLAKVYYPTDAKANSMANVQAVWSNYKYKGEGTVVSVIDS GIDPTHKDMRLSDDKDVKLTKSDVEKFTDTVKHGRYFNSKVPYGFNYADNNDTITDDKVDEQHGMHVAGIIGANGTGDDPAKSVVGVAPEAQLLAMKVFTNSDTSATTGSDTLVSAIEDSAKIGADVLNMSLGSDSGNQTLEDPEIAAVQNANESGTAAVISAGNSGTSGSATEGVNKDYYGLQDNEMVGTPGTSRGATTVASAENTDVITQAVTITDGTGLQLGPETIQLSSNDFTGSFDQKKFYVVKDASGNLSKGKVADYTADAKGKIAIVKRGELTFDDKQKYAQAAGAAGLIIVNNDGTATPVTSMALTTTFPTFGLSSVTGQKLVDWVTAHPDDSLGVKIALTLVPNQKYTEDKMSDFTSYGPVSNLSFKPDITAPGGNIWSTQNNNGYTNMSGTSMASPFIAGSQALLKQALNNKNNPFYAYYKQLKGTALTDFLKTVEMNTAQPINDINYNNVIVSPRRQGAGLVDVKAAIDALEKNPSTVVAENGYPAVELKDFTSTDKTFKLTFTNRTTHELTYQMDSNTDTNAVYTSATDPNSGVLYDKKIDGAAIKAGSNITVPAGKTAQIEFTLSLPKSFDQQQFVEGFLNFKGSDGSRLNLPYMGFFGDWN DGKIVDSLNGITYSPAGGNFGTVPLLTNKNTGTQYYGGMVIDADGNQTVDDQA1AFSSDKNALYNDISMKYYLLRNISNVQVDILDGQGNKVTTLSSSTNLTKTYYNAHSQQYIYYHAPAWDGTYYDQRDGNIKTADDGSYTYRISGVPEGGDKRQVFDVPFKLDSKAPTVRHVALSAKTKNGKTQYYLTAEVKDDLSGLDATKSVKTAINEVTNLDATFTDAGTTADGYTKIETPLSDEQAQALGNGDNSAELYLTDNASNATDQDASVQKPGSTSFDLIVNGSGIPDKISSTTTGYEANTQGGGTYTFSGTYPAAVDGTYTDAQ GKKHDLNTTYDAATNSFTASMPVTNADYAAQVDLYADKAHTQLLKHFDTKVRLTAPTFTDLKFNNGSDQTSEATIKVTGTVSADTKTVNVGDTVAALDAQHHFSVDVPVNYGDNTIKVIATDEDGNTTTEQKTITSSYDPDMLKNPVTFDQGVTFGSNEFNATSAKFYDPKTGIATITGKVKHPTTTLQVDGKQIPIKDDLTFSFTLDLGTLGQKPFGVVVGDTTQNKTFQEALTFILDAVAPTLSLDSSTDAPVYTNDPNFQITGTATDNAQYLSLSINGSSVASQYADININSGKPGHMAIDQPVKLLEGKNVLTVAVTDSEDNTTTKNITVYYEPKKTLAAPTVTPSTTEPAQTVTLTANAAATGETVQYSADGGKTYQDVPAAGVTITANGTFKFKSTDLYGNESPAVDYVVTNIKADDPAQLQAAKQALTNLIASAKTLSASGKYDDATTTALAAATQKAQTALDQTNASVDSLTGANRDLQTAINQLAAKLPADKKTSLLNQLQSVKDALGTDLGNQTDPSTGKTFTAALDDLVAQAQAGTQTDDQLQATLAKILDEVLAKLAEGIKAATPAEVGNAKDAATGKTWYADIADTLTSGQASADASDKLAHLQALQSL KTKVAAAVEADKTVGKGDDTTGTSDKGSGQGTPAPATGDTGKDKGDEGSQPSSGGNIPTNPATTTSTSAD DTTDRNGQHTTGTSDKGGGQGTPAPATGDTGKDKGDEGSQPSSGGNIPTNPATTTSTSADDTTDRNGQH TTGTSDKGGGQGTPAPATGDTGKDKGDEGSQPSSGGNIPTNPATTTSTSTDDTTDRNGQHTTGKGALPKT GETTERPAFGFLGVIVVILMGVLGLKRKQREE

TABLE 3 Exemplary Lactococcus lactis cremoris proteins notfound in Lactococcus lactis cremoris Strain A SEQ ID NO. name uniprot_idProtein Sequence 24 Cluster: J7TTI4MTQFTTELLNFLAQKQDIDEFFRTSLETAMNDLLQAE TransposaseLSAFLGYEPYDKVGYNSGNSRNGSYSRQFETKYGT VQLSIPRDRNGNFSPALLPAYGRRDDHLEEMVIKLYQTGVTTREISDIIERMYGHHYSPATISNISKATQENVATFHERSLEANYSVLFLDGTYLPLRRGTVSKECIHIAHL ALHQKDRRLFLDMKSPQMKTMLLGPPC 25Cluster: T0USG6 MQKRYSKEFKETLIVFYHSGQSVTQLSKEYDVAPATI TransposaseYKWIDLYSKSNESSVSKADFLELKRQLAKVKEERDIL KKY 26 Cluster: A0A1V0NYX4MTYNSTLPKVFVYLLTTIETLYQTRVPLEVQNRKNVH TransposaseLATSDCLVIACYLWGVLHFSETLKAKHQLAQSLFPNFLEYYRFVRRCNALLPSIQVIRQALVFKEVEGMSVSIIDSFPIPLCQPIRNFRSKVLGDYANVGYNATKGQYFYGCKCHALVSESGYVIDYTITPASMADSSMTEEVLNQF GTPTVLGDMGYLG 27 Cluster: T0UZJ0MISYHISKQPSYQSIDIALNQALAVTSDCPYRRTFHS TransposaseDQGWGYQMRDYVSKLKSHRIFQSMSRKGNCHDNS IS1077VMENFFGLLKQEIYYGHIFSSFEELEQVIVIWIRYYNT (Fragment) KRIKQKLNWMSPIQFRLNYQNN28 Cluster: S6EVX7 MPENKNFSRRSKKETGKKSLKIPKIRPKKQKNLKK Penicillin-binding protein 2A 29 Cluster: G8P734MKVTGFPKATYYYWVNCFERVNKDELIEKEMLKIRQ TransposaseEHANAGYRPMSELLKQRGYHVNHKKVQPLMKKLGL RVTSYWHKSRKYNSYKGNVGTVAKNKLHRRFRTSIPHQKITTDTTEFKYYEDGIQKKCYLNPYIDLFNSEVISYHISKQPSYQSIDIALNQALAVTSDCPYRRTFHSDQG WGYQMRDYVSKLKSHRIF 30 Cluster:A0A1V0PJ39 MAKNKLHRRFNTSIPHQKITTDTTEFKYYDKGVQKKL TransposaseYLTPYLDLFNNEVISYEISKQPTYQAIATALQEALELTSDCLYRRTFHSDQGWAYQMKNYVFKLKSQKIIQSM SRKGNCHDNSVMENFFGLLKQEIYYGHVFNSFEELEQAITKWIHYYNTKRIKKKLNWMSPIQYRLTYSK 31 Inner_ P75788MEHSATQRESQKIWTAIKNWFLVDKVFLISFIIAIIAISL membrane_GGVTTRFFNYHVIVTVSGLMLVIGGFKETGLLQYLGQ protein_TLVKRSTTTRQLVRFTTLLTFFLAVFFTNDLTILTVLPL YbiRYLAITKEIKNRKSVYIGAALIVPACHIGSALLPQGNPHNLYLYSFYKVAAHHGGVPLTNLDFFKGTGALWILGLLILMIACQFIDNEPLVIETKVNQFNKVETSIFVVLMLLMAASVFGYVNFYLAGAVVALVVLIYRPRLFKGIDYHLLFTFIFFFLIVGNIANIHVLTDFISNTLVGPQASFLGTVIMSQFISNIAAPILISPFTPHAVSLVLGADIGGIGTIVSSMATLIAYKVIRMNARGETRGFVKYFIIVNAGFVLILTLIGLIIV TLVG 32 Cluster: G8PA31MTYNSTLPKVFVYLLTTIDTLYQTRVPLEVQNRKNVH TransposaseLATSDCLVIACYLWGVLHFSETLKAKHQLAQSLFPNFLEYSRFVRRCNALLPSIQVIRQALVFKEVEGMSVSIIDSFPIPLCQPIRNFRSKVLGDYANVGYNATKGQYFYG CKCHALVTVNQAMS 33 Cluster:A0A1V0PFP4 MAQSLFPNFLEYYRFVRRCNALLPSIQVIRQALVFKE TransposaseVEGISVSIIDNFPIPLCQPIRNFRSKVLGDYANVGYNA for insertionTKGQYFYGCKCHALVTVNQAMS sequence element IS982B 34 Cluster: A0A0E2QIB2MARRKFDKQFKNSAVKLILEEGYSVKEVSQELEVHA Transposase NSLYRWV 35 Cluster:G0WKP8 MQSYDLLDELDSEDKFRKDIKYSRQLPEMFSTEDINA ReplicationASENITYAILGELRDRYNGSEPVTFSYQELAELGGLW proteinVTRKNGVKSLYNGKRLQKIMYDLNEALKNFSYYQVRETNDDGTPKSWKTINIFSVIDFDGTKKEVKLTISNAQISSEQVDAKGHVIDKPLYVYDLINSKDWRTVKHLQYNRGINNSLPSKYSKRVYRFISEFRSFPKGTKMRIDDFDKKILKILKTQEDSFNTKEVFDLRKNRKKYLETAVKEISELNTPEGTQIVKNLDYIYHTSGRRIQSIEFTYTPFNADLSGSNHISMNSRTSSPGTDSPFINEARMVLEYFNYLSKVNFNLDENGIIKHLPNYYDIQFELDDIQLLQPIHKLLESGVAIDELLQVAEMKAIDWKLDSNQMINNFRPSVVFGNKFSEYRAFLTTYKAQNIHKLVFDSSSDFYVPMNG PWDSK 36 Cluster: G0WKP9MTYNSTLPKVFVYLLTTIETLYQTKVPLEVQNRKNVH TransposaseLATSDCLVIACYLWGVLHFSETLKAKHQLAQSLFPNF for insertionLEYSRFVRRCNALLLSIQLIRQALVFKEFEGIDVSIIDS sequenceFPIPLCQPIRNFRSKVLGDYANIGYNATKGQYFYGCK elementCHALVSESGYVIDYVISPASIADSTMAEEVLSQFGTPI IS982BVLGDMGYLGQVLHDRLELKEIELITPVRMNMKKKDITFPNFSKRRKVIERVFSFLTNLGAERCKSRSSYGFLVK LEMTLLTYSLILKSAKTVNSMTLRYSTGYQVMAE37 Cluster: A0A0V8DWK8 MEKVTDEIKNVVQRLLDDDENFSGWYIEKELEKIGIKUncharacterized VSRMTISNLRNKKTTLGNTKFETLEGLYHFAKTHENI protein NKE 38Sporulation_ P37522 MKTISLLNLKGGVAKTTTGGNIAKGLANRGFKTLLIDT initiation_DMQANATSIFLEDKRSKEDYKGFAELIVDEKLDDVD inhibitor_QYVYNVSENLDMIGSSLAVAESELKVRNSFNRNSSNIVK protein_SojKVLKKLDSKYDYCIIDCAPTINLITLNIIIASDEIIIPIKIDKFALEGYRTTLKNINQIIDDYELDTEVTVLYTMVNRNNIDKQFIQEISGNRFETTIRHQAKPVTESALKNEVLID SSKSSKVKDDYLNLIDEIVKRG 39Nucleoid_ MF_02015 MSNSFGFTDLMNKDEHKRKKTNTKNIPIEEIKENENN occlusion_NYDLVDIDKLADSIDELGLLQPVLVKQRDKYSYELIAG proteinHRRFNAIKKLISENRLPEDYEVLAKKVDEDEDELVTRLKLHETNLQTRSLLKMPEEEKIAIIDDYMDILDKAKKQGLQINGKPVKGKTRDLIAERFGISHYTAQKLIRKAKEQGGEEEGAKISPQKKTAKKPITQLKKIETQLEKLEFE GTEEEQEIKKKLIELLMK 40 Cluster:Q2VHI9 MSVDRSYSPYEVIRAYHDRGMMKWGAFATGELTEA UncharacterizedQNTFEKEKKDDKVIQTLPHHVVLHLLNQSFSNQVQIK proteinVKYQSKDKLTEVYGFVSEFINNQVRVKSTDKIYLISIE QIINIS 41 DNA_ P9WNT3MEQLKLNKYFDYSLEPRRAILFQDVKSNYASIECVQR polymerase_NLNPLTTSLCVMSRADHSKGLTLASSPTFKKVFGMK IV_1NVSRASDLPFLIETRKFNYPQWYRTHTDIHGQRTEPTLQYVAFIESWAKRTWIVPPQMQLYVDYKIEVTDILTNYTSIDEIHSYSIDESFLDITESLNFFYPEIKNRYEQMNRIALDLQREIRDKLGLYVTVGMGDNPLLAKLAMDNYAKHNDNMRALIRYEDVPNKLWTIPKMTDFWGIGKRTEKRLNKLGITSIKELANADPLLLKQKLGTIGLQHFFHANGIDESNVREKYTPKSTSFSNSQILPRDYHKQREIELVIKEMAENLAIRLRKGGKLASNLSLYAGAASTSEYSSVKVSRNIEATQNTKELQDLAISLFREKYQGGAIRQIGISGNQLSDSSVKQLSLFESVQENQTNKKQESLQKAIDEIRETFDFLSIQKASSLSEGSRVIYRNKLIGGHAASQDK EEKDVS 42 Cluster: G8P9Y4MDKYIRRAYQRMNQMSFGGQALAWFLSIRLSDLVLK Uncharacterized K protein 43Cluster: A0A1V0P5K1 MAEAKFEAALIKKLEAEGWTYRKDLSYVSIKVLEGH HsdR type IWREVLNENNAYKLNGKPLSDVEFGLVIQEVQRIKTP restrictionYDAQLLLVGAGGVGSIPITRDDGSNLEVWTNVKYLD enzyme R TK protein 44 Cluster:G8P9Y1 MIFKLRNRTEIAINKRKPKEPIIFHSDHGSHFKSASFR TransposaseKLLDEHQLLASYSKPGYPYGNAVTEVFFKYLKHREIN TnpARRTYHSIQEVQLSCFEYIEQFYNNYNPHSANNGLTP N 45 nanMKVTGFPKATYYYWVNCFERVNKDELVEKEMLKIRQ EHANAGYRPMSELLKQRGYHVNNKKVQRLMKKLGLRVTSYWHKSRKYNSYKGNVGTVAKNKLHRRFRTSIPHQKITTDTTEFKYYEDGIQKKCYLNPYIDLFNSEVISYHISKHPSYQSIETALNQALAVTSDCPYRRTFHSDQG WGYQMRDYVSKLKSHRIFQSMSRKGNCHDNSVMENFFGLLKQEIYYGHIFSSFEELEQVIVIWIRYYNTKRIK QKLNWMSPIQFRLNYQNN 46 nanMVKYSIELKQRVIQDYLSGKGGSTYLAKLHNVGSSS QVRRWIRNYRAEGLHTAHSKVNKNYSMELKENAVQCYLTTDLTYEAVARKFEITNFTLLASWVNHFKIYGEVPISKKRGRRKKLESIASSMTQNPNDSQRIKELEQELRYAQIEVAYLKGLRRLEKNALMNKNQDSSTVSVKPSN SKKS 47 nanMKHHGKIKIKHAVKVLKVSRSGFYEYMHRRPSKQQVEREILSEKIKAVFHEHKGRYGAVRITKVLHNTGIMTNTKRVGKLMHLMGLYAKGSRYKYKHYNRKGASLSRPNLINQIFKATAPNKVWLGDMTYIPTKEGTLYLAVNIDVFSRKIVGWSMSSRMQDKLVRDCFLQACGKEHPQPGL IVHTDQGSQYTSSRYQSTLRQVGAQSSMSRKGNPYDNAMMESFYKTLKRELINDAHFETRAEATQEIFKYIE TYYNTKRMHSGLDYKSPKDFEKYNS 48Cluster: A0A1V0PDS7 MNHFKGKQFKKDVIIVAVGYYLRYNLSYREVQELLYD TransposaseRGINVCHTTIYRWVQEYSKVLYDLWKKKNRQSFYSWKMDETYIKIKGRGHYLYRAIDADGLTLDIWLRKKRDTQAAYAFLKRLHNQFGEPKAIVTDKAPSLGSAFRKLQSVGLYTKTEHRTVKYLNNLIEQDHRPIKRRNKFYQSLRTASSTIKGMETLRGIYKKNRRNGTLCSDPQNIDFSNLYFWKGYNKLDTKLREIVERFIMARRKFDKQFKNSAVKLILEEGYSVKEVSQELEVHANSLYRWVQEVEEYGESAFPGNGTALADAQHKIKLLEKENRYLQEELELLK KFRVFLKRSK tRNA-Met nan RNA 49Cluster: D2BMP1 MKTGDKITLSNGEQATVVSGDINLYKYALIVELENHD Cold-shockVRVVDRETLTLAKENPHENLGNHKKINKF protein 50 Cold_shock- P0A355MNKGTINWFNADKGYGFIMADDMQDVFAYLLSIQGN like_protein_DFKKYDEGQKVTFDIKMTSRGRYASNVHKR CspLA 51 Cold_shock_ P96349MANGTVKWFNADKGFGFITSEEGKDLFAHFSAIQSD protein_2GFKTLDEGQKVEFDVEEGQRGPQAVNITKA 52 Cluster: Q2VHI5MARRKFDKQFKNSAVKLILEEGYSVKEVSQELEVHA UncharacterizedNSLYRWVQEVEEYGESAFPGNGTALADAQHKIKLLE protein KENRYLQEELELLKKFRVFLKRSK 53nan MKHHGKIKIKHAVKVLKVSRSGFYEYMHRRPSKQQVEREILSEKIKAVFHEHKGRYGAVRITKVLHNTGIMTNTKRVGKLMHLMGLYAKGSRYKYKHYNRKGASLSRPNLINQIFKATAPNKVWLGDMTYIPTKEGTLYLAVNIDVFSLKIVGWSMSSRMQDKLVRDCFLQACGKEHPQPGL IVHTDQGSQYTSSRYQSTLRQVGAQSSMSRKGNPYENAMMESFYKTLKRELINDAHFETRAEATQEIFKYIET YYNTKRMHSGLDYKSPKDFEKYNS 54Cluster: A0A0V8EKB0 MLENEYFVFTSTLTTMIRKQAQSIITGLKGHNQNSVT DNA-KNTTRLVTGYFPIDLIKGYRPSQKLSEAKQAEQRGQ directed DNAQIIMMTEKQFIDFLAQSFYLLSQGL polymerase 55 Cluster: A0A0V8EK89MKLREIIKEIPDDDWLEIIEQSSINYRSFIGRAPKKYIV UncharacterizedGELLDYEALYIGEVKKNKNYQNHRFLVEDKFIEHSGR protein 56 Cluster: A0A1V0PE50MKKTIIFILHIPFILLLWLCITSPFFIKNSLLNSSFGHIFK UncharacterizedGVENISHSGPLATVLLLFVIPLLSLISCLYLAFKKNQSG proteinRKYVIYILMSLFSLVCLSVFSVIMIIGLGNYL 57 nanMNHFKGKQFKKDVIIVAVGYYLRYNLSYREVQELLYDCGINVCHTTIYRWVQEYSKVLYDLCKKKNRQSFYSWKMDESYIKIKGRGHYLYRAIDADGLTLDIWLRKKRDTQAAYAFLKRLHKQFGEPKAIVTDKAPSLGSAFRKLQSVGLYTKTEHRTVNYLNNLIEQDHRPIKRRNKFYQSLRTASSTIKGMETLRGIYKKNRRNGTLFGFSVSTEIKV LMGITA 58 Cluster: G8P9W7MKTQELNLKQFVMLSEKELQEISGGGGWGSAFAGW UncharacterizedLGGIGVNSGQTAQQVVNQLNGVTDFHAYNHNPYGS protein GGTPND 59 Cluster: Q2VHK7MFTLIFSNLTGGIIIKAIYKDKTVDVWEISKNNEQPDW UncharacterizedVKNAFKENYLSWYDERLKILMNGIKPSAKSSLKLGIM proteinGSVAGSLAGGLAGNNIYVMGEIGDYLDITNRKVVSK EKFLKKYSV 60 Cluster: Q2VHK6MKYFVTFLSPTQNMGILNWQTMILDDYLVDDSYWEN UncharacterizedTKLELSKEVEWITQSELYKKVKRNDGSGNDIILSVPV protein SAVLETIKSFFILGHS 61Transcriptional_ Q3J6K8 MRNTKEKILTATEQUYKKGYTGTSINDILDETATGKGregulator_AcuR QFYYYFDSKKEACLAVIDNHVKIWQKHLLNGILSRDESPLANLKEMLDWIYSDHAQKKIYYGCPVGNLVIELSALDEDFRKPLEQLFSDLQKKIAENLSGLTGLLVKQNLPAAHAIIAQIQGSLLLLKVTQDLNVLESNFDLLKTIFEKV GEK 62 Cluster: Q2VHK4MKKLDMIVIGPGPAPPTAVIRRKSLCQQLNLKKKSKL UncharacterizedLQVDAIRREYTIADVQKRWQSCQTFIDVLRKGILKQLI protein AELS 63 Cluster:A0A0D6E0F2 MQNNYTSKGKHLTESERLLIERWHNKEKVSNREIAY TransposaseRLGKAPQTIHNEIQRGTVQLKYKTKYSAKIAQESYKTLRTHSKRSTKLNAQLDDQISKAVKNKISLEVIHQELKGVVCLRTLYNWISSGILSVAYHELLYPQYRKPKKQRVTQPKHMLGQSIEERPESVDERSEYGHWEIDTVLLTKEKGECLLTLTERKTRLEIIRLIPNKTTHSVNQALRGIEFLALSVTSDNGREFAKLSEALDCPVYYCHAYASHERGTNENHNRMIRRHLPKGTKKTTKQVVAYIENWMNNYP RKMFNFKTPNQMLIESI 64Isochorismatase_ P0ADI7 MFNNKNTAFVVTDPQVEFLKPKGAGYGLTKDILRKYfamily_protein_ HTTENLTELFKHAKAKGYKIFISPHYFYDHDQNWKFG YecDGQGEQMMLNNKMFHREHQYQETVKGSGADFVEELKPYLDENTIITSPHKIFGPESNDLALQLRKNGIDTVILAGMNANLCVDSHLRELVESGFHVHVAADATGAPGQE AYDAAITNFGFVADRTMSTAKVLEEL 65putative_ P77212 MKKIDVKNIVVGFGKGGKTLAKFLSGKGESVVVIEQS pyridine_TLMYGGTCINIGCIPSKFLIVNGEKGLKFTEASEKKAM nucleotide-LTGNLNLKNYHMIADEATAEVIDGKAKFVSDHEIEVM disulfideDAEGEVIAQLIGERIFINTGATPVLPPIPGLVDSRNVVTSTELMDLKQLPEHLTIIGSGYIGLEFASMFASYGSKVTVLDIFDNFLPRDDEDISKLVRSDLESRGIIFKLGVKIDAITDNSVEIINKEGKKVSILSDKILVATGRKPNTAGLGLENTNIQLGQRGEIVVNDKLETTVQNVWALGDVHGGLQFTYTSLDDFRIVSNNLYGDGKRSLSDRKNVPTSVFITPALSKVGLNEKDAKAAGIDYRLFKLAATAIPKSAVLNQSKGLLKALVDPETDKILGITIYAEESYETINLVSLAIEVGLPYTLLRDKIYTHPTMTEALNDLFAAKNEVK 66 nanMELKENAVQCYLTTDLTYEAVARKFEITNFTLLASWVNHFKIYGEVPISKKRGWRKKLESIASSMTQNPNDSQRIKEPEQELRYAQIEVAYLKGLRRLEKNALMNKNQD SSTVSVKPSNSKKS 67 Cluster: G8P734MKVTGFPKATYYYWVNCFERVNKDELIEKEMLKIRQ TransposaseEHANAGYRPMSELLKQRGYHVNHKKVQPLMKKLGL RVTSYWHKSRKYNSYKGNVGTVAKNKLHRRFRTSIPHQKITTDTTEFKYYEDGIQKKCYLNPYIDLFNSEVISYHISKHPSYQSIDIALNQALAVTSDCPYRRTFHSDQG WGYQMRDYVSKLKSHRIF 68 Cluster:I7LSK3 MSHKGNCQDNSVMENFFGLLKQEIYYGHIFSSFEEL TransposaseEQVIVIWIRYYNTKRIKQKLNWMSPIQFRLNYQDN 69 nanMTYNSTLPKVFVYLLTTIETLYQTRVPLEVQNRKNVHLATSDCLVIACYLWGVLHFSETLKAKHQLAQSLFPNFLEYSRFVRRCNALLPIIQVIRQALVFKEVEGMSVSIIDSFPIPLCQPIRNFRSKVLGDYANVGYNATKGQYFYGCKCHALVSESGYVIDYTITPASMADSSMTEEVLSQFGTPTVLGDMGYLGQSLHDRLELKEIDLMTPVRKNMKQKKILFPNFSKRRKVIERVFSFLTNLGAERCKSRSPQGFQLKLEMILLAYSLLLKSAKSLEPETLRYSIGYQVMA K 70 Putative_ Q5XD45MIKLVAIDLDGTLLDPNRQITAEVKTAVKKAKAAGVKI phosphataseVITTGRPLPGVVDILKALELTDQSDYVITYNGGLVQQATGEEFIKETLSSEDWLDLDAAARKIGLPIHAITREGIYTPNHDVGRYTVQEAQMVKMPLYIRQPEDIAALEIAKVMMVDEPAALDDGIAYLPFEFFERYNVVKSTPFYLEFMNKKASKGSAVQHLAEKLSFDLDEVMAIGDEENDRSMLEVAVCSVVMENGKSKLKKIAKYVTKSNAKSGVAY AINEWVLKDYQD 71 nanMKITFDEKTADKIKAFGDVDLVFDFDHTLSEVNTEVDACAGGISRYRIVAVEKGNVPEVFDASIDSEFGPIYYKGYGSYFFQDEMYTKINPSYNLIELHSTAELLSPNLLIV DFRGKQKAS 72 Cluster: T0SFW4MLSAGLLGIDPGHYIEHAFIGLVADKLRSFDLGVKIYE Uncharacterized SQEKTNPFYDIprotein 73 Cluster: IS30 Q47803 MTYTHLTSNELAMIEAYYNNHQSVAKTAVLLNRSRQfamily TIHKVYQFFKTGHNALDYFNQYKKNKTRCGRRPIVLS transposaseDEQTEYIQKRVVQGWTPDVIVGRAEFSISCSMRTLY IS1062RMFKQGVFEVTHLPMKGKRKANGHKETRGKQSFR RSLRDRGNDYSKFNQEFGHLEGDTIVGKKHKSAVITLVERLSKVIITLQPEGRRAIDIENRLNQWMQSVPKHLFKSMTFDCGKEFSNWKSISNINDIDIYFADPGTPSQRGLNENSNGLLRKDGLPKQMDFNEVDESFIQSIASKR NNIPRKSLNYKTPIEVFLSHICKEELSNLI 74Cold_shock- P0A355 MANGTVKWFNATKGFGFITSEDGQDLFAHFSSIQSD like_protein_GFKSLDEGQKVEFDVEEGQRGPQAVNITKA CspLA 75 nanMNYFKGKQFQKDVIIVAVGYYLRYNLSYREIQELLYDLGINVCHTTIYRWVQEYSKVLYHLWKKKNRPSFYSWKMDETYIKIKGRWHYLYRAIDADGLTLDIWLRKKRDTQAAYAFLKRLHKQFGQPRVIVTDKAPSIGSAFRKLQSNGLYTKTEHRTVKYLNNLIEQDHRPIKRRNKFYRSLRTASTTIKGMETIRGIYKKNRRNGTLFGFSVSTEIKVLM GILA 76 Cluster: G0WKN9MVTYTDLLPKPTENQQAFILDHGKTEDDGQLKYADD PIL7_11AKSYGWNMRQYGKLKAGAVVLNRHPGKITKDRKWEIYGGGYVESVSDEDENGNVTAVITHAFTIEPPIKQGDSFIENFDWNTPNKKKRKKPNSWAYFWDQYGMNEISYTDFVGLIENRHLSPIDDTQSLPVEKDLTNAEVEEIEEASSKGFTVLVDEVGPNRPNGTQKRKFTGRHTDWERVNKAKQKTGALGEEIVLDFLIQKAEKNKTKLPEHVSKTEGDGHGYDIRAFDQSGNEIHIEVKASKTNFSDGFEMSANEVASSLEDTPYKIYFVHDLDVTSKVCKIKIYDG PFTEENFMMVPTNYKIFKK 77 Cluster:Q2VHL9 MFWTNVKYLDAHILKQNEQLKYENPTEENKLKIKALQ UncharacterizedLERKDLQAQYRKVIKKMKTYDAGQEIVQEKLKEKEIN protein KEKTQDIPS 78 Cluster:S6EPX4 MIYTIGYYIAVIGLVIMMFGFKSFYSQMNKWSRFGFIF UncharacterizedLALGLAFPIVYDFIVGFINGLLKNVN protein 79 Aminodeoxy P28819MKLLLIDNYDSFTYLLVQYFEELDCSVTVVNDQDKM chorismate/SQKIRISPDFICENYDAITISPGPKTPKEAVFSRDVVQ anthranilate_LYAGKIPMLGICLGQQVIAECFGGNVVLGERPMHGKI synthaseSVIRHNCQGIFKGLPQNLKVARYHSLIVDKLPNDFEIDAQSEDGVIQAMHQPKLKLWALQFHPESLVTEYGHE MLNNFLKVV 80 Aminodeoxy P05041MKEFIIKNTDIWKIFLKYYRSDEEIVFLHSSQATENEH chorismate_YSILAHKPYKKVSKYKGQVFFNGEKKKFNFLDAVDLL synthase_KNEKVERPKNWPFYPELLGFVSYEQDPAYFAAYDE component_1VLLFDHRTKRLRVVQFEQTDGQYWLTESEEIEVDSEIEFDGQNGIGAVFIDQTRQEYIASIKRLQDYMKAGDIYVANLTQQFEIWSDQKPIDVFKKTRNQIPAPFSSFLQYPEWKMTQISSSVERFVSIHDGALISKPIKGTIARGEDVVTDRLQKEILSNSIKERTELLMVTDLLRNDIARISQPFSLSVPKFAEIETFSHVHQLVTSIKSRIKEDLTFSEFMTALFPGGSITGTPKKRAMEIIKEVEKQPRGIYTGMQGWLSREMDLDMNIVIRTLVHDGEHYQLGVGGGITFESEAEAEFSEILLKAKPFLDILGLKDVPSILFTTGLVKNGELLNLEGHVNRLKKQYHHPDLEEKLRKFAQNVTDGVLRVSTDGDSLNPEIRQLTHSNESYRVKLSSINDKPSPLSNFKLSGPDFQKVFRQEVLDVKKEGFQDILFHTDGLVSELSIGNFVAKKGNQYETPAKYALKGTFLDLFAKNHTLIYKDIAISDLKNYDCFYMTNAVRGLVEIKIDGISGSV AKFSKKSILV 81 nanMNYFKGKQFQKDVIIVAVGYYLRYNLSYREIQELLYDRGINVCHTTIYRWVQEYSKVLYHLWKKKNRQSFYSWKMDETYIKIKGRWHYLYRAIDADGLTLDIWLRKKRDTQAAYAFLKRLHKQFGQPRVIVTDKAPSIGSAFRKLQSNGLYTKTEHRTVKYLNNLIEQDHRPIKRRNKFYRSLRTASTTIKGMETIRGIYKKNRRNGTLFGFSVSTEIKVL MGILA 82 Cluster: Q8GAR6MDNKDIELIQQMENKYDTFMPVLTNLIDSVEKFNSIY UncharacterizedNNYIELRNFYGSEKWFEYMEIEKIPVKCGVLTEDQLF protein DMISDHNELLGVLLDLTSKMYKNF83 Cluster: Q7BLP2 MVQDTLLDSFRAGRRNYTIFQVGKATLLRVSDVMKL IntegraseKKTDVFNLDGTVKQTAFIHDQKTGKGNTLYLKPVQQDLMLYHAWLIQQNMNSEWLFPSTSRPYRPITEKQFYKIMARVGDLLGINYLGTHTMRKTGAYRVYTQSNYYW LSYAFIKPFK 84 nanMNHFKGKQFQQDVIIVAVGYYLRYNLSYREVQELLYDRGINVCHTTIYRWVKEYSKILYHLWKKKNKQSFYSWKMDETYIKIKGRWHYLYRAIDADGLTLDIWLRKKRDTQAAYAFLKRLHKQFGQPRVIVTDKAPSIGSAFRKLQRNGLYTKTEHRTVKYLNNLIEQDHRPIKRRNKFYRSLRTASSTIKGMETIRGIYKKNRRNGTLFGFSVSTEIKIL MGIPA 85 Bis(5′- MF_00199MYNEVFVVSDIHGEYKKFKEILKYWDSNRQQLILLGD nucleosyl)-LCDRGLQSYECFYLAKYLCDNYGAILIKGNHEDLFLK tetraphosphatase,_FLNKTEDFKENYIKNGGLKTLESFGYSENNTFKDIVL symmetricalDIKKNNDKLIEFLTYLPNFYEWNDYIFVHAGVNLKINNWKDTSIRDFMWIREDFHFTPNRLNKTIVFGHTETKILNKNNKYDIWIHDNKIGIDGGAVYGGYLYGVILDVHGI KDYVYV 86 Cluster: T0VLA4MINYQGEVFTETEFYGREILEAIQLTNKFPTPKKVLID UncharacterizedMLEEMIHEQLDLIDKEELNNYINAKKYVQTLTEDEVK protein NLCFEVKDLYEDVLKEFEIKL 87Cluster: T0V569 MKKTGITNFSVFARRACCNKEIFTLDFSEYKNIISEISA Molybdopterin-TKSELKRIGNNINQIAKHLNENKNNQTESLMSDYQN guanine QLESLEEKIQKVVHYISEGdinucleotide biosynthesis protein MobC 88 Cluster: Q9FB66MTVIYMPKQSNGTVHSAKDLKQLIDYVMNSEKTNDF RelaxaseEYVSGQNILDIHSTCDEMLATRTMANALKNKPQKNE Mob DEIRFGYHFVQSFSPDDHLTPEQVHEIGCKTMKEYLGSS AEFIIATHTDKPHLHNVRPDRVLSQVV 89Group_II_ P0A3U0 MKPTMAILERISKNSQENIDEVFTRLYRYLLRPDIYYV intron-AYQNLYSNKGASTKGILDDTADGFSEEKIKKIIQSLKD encoded_GTYYPQPVRRMYIAKKNSKKMRPLGIPTFTDKLIQEA protein_VRIILESIYEPVFEDVSHGFRPQRSCHTALKTIKREFG LtrAGARWFVEGDIKGCFDNIDHVTLIGLINLKIKDMKMSQLIYKFLKAGYLENWQYHKTYSGTPQGGILSPLLANIYLHELDKFVLQLKMKFDRESPERITPEYRELHNEIKRISHRLKKLEGEEKAKVLLEYQEKRKRLPTLPCTSQTNKVLKYVRYADDFIISVKGSKEDCQWIKEQLKLFIHNKLKMELSEEKTLITHSSQPARFLGYDIRVRRSGTIKRSGKVKKRTLNGSVELLIPLQDKIRQFIFDKKIAIQKKDSSWFPVHRKYLIRSTDLEIITIYNSELRGICNYYGLASNFNQLNYFAYLMEYSCLKTIASKHKGTLSKTISMFKDGSGSWGIPYEIKQGKQRRYFANFSECKSPYQFTDKISQAPVLYGYARNTLENRLKAKCCELCGTSDENTSYEIHHVNKVKNLKGKEKWEMAMIAKQRKTLVVCFHCHRHVIH KHK 90 Cluster: T0UZ98MKEISDNISKEYGCKIIVRPEQKLGNSHKNYLVYLAK PrimosomeNSYRKEIKNKLDFLMNHSHTWEDFKEKARALNLKVD assemblyDTKKYTTYLLEGSEQTKKIRDRSLKNDKFLKENLKER protein PriAIEKNTIGYSVDEVVKLWKDKESIQEKGREKEIEILLEHWQVTKETEKDLVVTIDTAFDNEATIKIPARCVDKLENGQYKIFIKKGDRFSYLDKKSPANHKIMYGATVAKNLQRQSGNIPLYSDNVNIKLKQVFHEFDFLISQGLSFDRSFETIGEELKATYQETQHQLDKLDTKILEYVETTKTLPYEDTSIRDTIKNLTKERDDLRDTLYKVDKNIQYYQKSE QRLEAYQKNQSPKHKARDDDFEI 91Cluster: A0A1B1RSI6 MSKNVKTIKELADELGTNKTRISRIINKNSIPTQKIKNKIReplication- VLEDNSVSLIRQYFKNETVSILRTELDKAHSHIEKLSN associatedLSDQQQRLALQDKKLLEEYKAENDSLKALKMPTEGS proteinQAEQANSQPKEEVKALKFEIRALQEELNKQKIHSQE RepX, RepB EREKLKAELTTPKKWYQFWKfamily 92 Cluster: G0WJT7 MSGFKRYDEDFKQSLVNLYQTGKTQTELCKDYGVSTransposase SSALAKWIKQYSQVRLEDNTVLTAKQIQELQKRNAQ LEEENLILKKASAIFMQNSK 93Cluster: F9VEW3 MKAKKRIGTRAFKIILLRDYGVNISEGRILRLLKSMTLP TransposaseKMSTIKPRFKSNKSPVFSSDNLLKQEFNPNSPNQVWTTDFTYISIGPKRHVYLCAILDLYSRKCIAWKVSDKIDAQLACDTLEIALNKRKPKEPIIFHSDQGSQFKSASFRKLLDEHQLLASYSKPGYPYDNAVTEVFFKYLKQREINRRTYHSIQEVQLSCFEYIEQFYNNYNPHSANNGLTP N 94 Cluster: G0WKP7MRQLADALNVSFEYLTDTEILPIYQELSDDNKQQTIN PutativeYAEDKLKSQKEQENIIHFRNSLIPYKQATEQALSAGL transcriptionalGEGYTDNIETCTVYWDKQVNYDIGIPIKGDSMEPEFH regulatorYGQTALIKYQSSPDYDGQVCAVDNVSMGNGFIKCVTVEEDGLLLQSLNIEEGQNGERKFPDIKLYWDDNPRII GKVVAAFTPIEIDFLFKNLEL 95 Cluster:A0A218PFY7 MERKKKKKENIWAIIVPILIIISLIGAWAYALRDSLIPND ExopolysaccharideYTKTNSSDQPTKTSVSNGYVEQKGVEAAVGSIALVD biosynthesisDAGVPEWVKVPSKVNLDKFTDLSTNNITIYRINNPEV protein EpsLLKTVTNRTDQRMKMSEVIAKYPNALIMNASAFDMQTGQVAGFQINNGKLIQDWSPGTTTQYAFVINKDGSCKIYDSSTPASTIIKNGGQQAYDFGTAIIRDGKIQPSDGSVDWKIHIFIANDKDNNLYAILSDTNAGYDNIMKSVSNLKLQNMLLLDSGGSSQLSVNGKTIVASQDDRAVPDYI VMK 96 hypothetical_MAQTIQTLALNVRLSCQLLDVPESSYYERINRHPSKT proteinQLRRQYLSLKISQLFNANRGIYGAPKIHHLLLKQGEKVGLKLVQKLMKQLQLKSVVIKKFKPGYSLSDHINRKNLIQTEPTKKNKVWSTDITYIPTQQGWAYLSTIMDRYTKKVIAWDLGKRMTVELVQRTLNKAIKSQDYPEAVILHSDQGSQYTSLEYEELLKYYGMTHSFSRRGYPYHNA SLESWHGHLKREWVYQFKYKNFEEAYQSIFWYIEAFYNSKRIHQSLGYLTPNQFEKVSA 97 Cluster: A0A0M2ZR43MVDAYLDNNLGDDLMIRYFASYFYQHKIYLVESREHI PolysaccharideRKTFYDIPNIYFYSEEDYKMNEYDFQLHVTIGGSMFIL pyruvylDDFKKLIRFRHRIKNSRKIKKRNIPSAIIGCNLGPFDKR transferaseNFGLKLAKFELKYKNLVTVRDKQSKELLLRGFKRKKI CsaB, csaBNIKLFPDIIFSKVLYKSIPKYGLGMTLSQVFRVTNVEF 98 putative_ P71059MKNKFSIIVPVYNGESHIKKCIDTLLKQTYNDFEIIIIND glycosyltransferase_GSTDDTKSVLTKFYAKNLKVKIVNTSNKGVSFARNLG EpsJINQSSGQYLLFVDSDDELSINALKYLSIMLNKKDRDLILFGFSLTGDNNRKNDTSILKSIANQNTDCKMNILKSILSTKNNILGYVWRAVYSLDFIKKNNIFFETHLKISEDYLFLLQSVEHSNNLFVITEEFYKYNLGETSMSNKFVPTLLNDMVWVNNWIESNILTVYPQFFVGFNCLVANTYIRYVQNAIRNKEENFMLKYREIKINKRKYNFQRSINQVIFHLDKFDFKSKIGVILFRIHLDIVYELLFNIKERKN 99 Cluster: Q3ZK44MTNLNRKKFFINFQSLVFFILIIIYGLTTKNVMGGSGIF EpsHSIDSILKYGILFICISVEGYIFLKNGNERRETSENYNNFKYYFIIITFLSLFASFKQVHFSFRTVQSFIFIFIPMLYSYLILNNWTFRQINFSMKIALFLSVIEYLFSIRMGFSQIISSLASINYNNTNASALESSTFALLSLGFAAYFGYYKKNFLCKIVSLLFVIMTFKRVITLSGCILVILGILKIKNLRVNRFFLIVSTITLVSFSLIYYYSIQPQNILEISEKIGFSIRDFSTNRTDRLAWLSMTDFKSYGLGSTTDFMYKLWGVDLEMDIVQLILEVGAFGVIVFIYFYLRFSKSNLYAFSFMALLLLNSILSSGMMSTFSWIIILIAMSTIMEYKEGM 100 Cluster: A0A0M2ZW08MKKLKISVIIRTYNEVKHIGEVLKSLTDQTYLNHEIIIVD TransferaseSGSVDGTLDIIERYPVKLVSINKEDFNYSYASNVGVQ 2,NSSGDIVCFLSGHSVPVYKNYLEKINEIFQETEIGACY rSAM/selenoGEVIALPDGSITEKIFNRIGYLKSKLSLNNKRFFLENKI domain-HPGIFSCSNACARKKLLLKYPFKVELGHGGEDVEVA associatedYRIIQDGYFVAKSVELLVMHSHGSSLKKFIKEYKAWG KMYEDVLKFIKKNNDKSQ 101 Cluster:Q3ZK46 MIFVTVGTHEQPFNRLIQKIDELVRDGQIKDDVFMQI EpsFGYSTYEPKYTKWASVIGYNDMTAYFNKADIVITHGGPSTYMQVLQHGKIPIVVPRQEKFGEHINDHQLRVSKQVIKKGYPLILCEDVSALKICIESSRIRTDEFIKSNNKN FISNFKKIIAFEE 102 Cluster:Q9X491 MKIALVGSSGGHLTHLYLLKKFWENEDRFWVTFDKT EpsEDAKSILKEERFYPCYYPTNRNVKNTIKNTILAFKILRKEKPDLIISSGAAVAVPFFWIGKLFGAKTVYIEIFDRIDKPTLTGKLVYPVTDKFIVQWEELKKVYPKAINLGGIF 103 putative_ P71062MEFFEDASSPESEEPKLVELKNFSYRELIIKRAIDILG sugar_GLAGSVLFLIAAALLYVPYKMSSKKDQGPMFYKQKR transferase_YGKNGKIFYILKFRTMIFNAEQYLELNPDVKAAYHAN EpsLGNKLENDPRVTKIGSFIRRHSIDELPQFINVLKGDMALVGPRPILLFEAKEYGERLSYLLMCKPGITGYWTTHGRSKVLFPQRADLELYYLQYHSTKNDIKLLSLTIVQSIN GSDAY 104 Tyrosine- P96717MIDIHCHILPGIDDGAKTSGDTLTMLKSAIDEGITTITA protein_TPHHNPQFNNESPLILKKVKEVQNIIDEHQLPIEVLPG phosphatase_QEVRIYGDLLKEFSEGKLLTAAGTSSYILIEFPSNHVP YwqEAYAKELFYNIKLEGLQPILVHPERNSGIIENPDILFDFIEQGVLSQITASSVTGHFGKKIQKLSFKMIENHLTHFVASDAHNVISRAFKMKEAFEIIEDSYGSDVSRMFQNNA ESVILNESFYQEKPTKIKTKKLLGLF 105Tyrosine- P96716 MAKNKRSIDNNRYIITSVNPQSPISEQYRTIRTTIDFK protein_MADQGIKSFLVTSSEAAAGKSTVSANIAVAFAQQGK kinase_KVLLIDGDLRKPTVNITFKVQNRVGLTNILMHQSSIED YwqDAIQGTRLSENLTIITSGPIPPNPSELLASSAMKNLIDSVSDFFDVVLIDTPPLSAVTDAQILSSYVGGVVLVVRAYETKKESLAKTKKMLEQVNANILGVVLHGVDSSDSPS YYYYGVE 106 putative_ P96715MQETQEQTIDLRGIFKIIRKRLSLILFSALIVTILGSIYTF capsular_FIASPVYTASTQLVVKLPNSDNSDAYAGQVSGNIQM polysaccharide_ANTINQVIVSPVILDKVQSNLNLSDDSFQKQVTAANQ biosynthesisTNSQVITLTVKYSNPYIAQKIADETAKIFSSDAAKLLNVTNVNILSKAKAQTTPISPKPKLYLAISVIAGLVLGLAIALLKELFDNKINKEEDIEALGLTVLGVTSLCSNE 107 Cluster: A9QSJ2MMKKGIFVITIVISIALIIGGFYSYNSRINNLSKADKGKE PolysaccharideVVKNSSEKNQIDLTYKKYYKNLPKSVQNKIDDISSKN biosynthesisKEVTLTCIWQSDSVISEQFQQNLQKYYGNKFWNIKNI proteinTYNGETSEQLLAEKVQNQVLATNPDVVLYEAPLFNDNONIEATASWTSNEQLITNLASTGAEVIVQPSPPIYGGVVYPVQEEQFKQSLSTKYPYIDYWASYPDKNSDE MKGLFSDDGVYRTLNASGNKVWLDYITKYFTAN108 Cluster: O06027 MNNLFYHRLKELVESSGKSANQIERELGYPRNSLNN EpsRYKLGGEPSGTRLIGLSEYFNVSPKYLMGIIDEPNDSS AINLFKTLTQEEKKEMFIICQKWLFLEYQIEL109 hypothetical_ MSVSIIDSFPIPLCQPIRNFRSKGLGDYANVGYNATK proteinGQYFYGCKCHALVSESGYVIDYTITPASMADSSMTEEVLSQFGTPTVLGDMGYLGQSLHDRLELKGIDLMTPVRKNMKQKKILFPNFSKRRKVIERVFSFLTNLGAERCKSRSPQGFQLKLEMILLAYSLLLKSAKSLEPETLRYSI GYQVMAK 110 Cluster: A0A0B8QXZ2MTIKNKKDLSSSIEQLEKAINQQETILKKFDNEQLDFE SignalQIKKLENLLIQEREKAKQVQIKINRSVLQNNSENYKER transductionKKRTRQLIQKGALLEKYLEAKHLTVDETEQLLQIFAN histidine MINKPELLVNFIGK kinase111 Cluster: B1N0G0 MVQQIVLPIKDSNILKMVQDTLLDSFRAGRRNYTIFQ TyrosineVGKATLLRVSDVMKLKKTDVFNSDGTVKQTAFIHDQ recombinaseKTGKANTLYLKPVQQDLVVYHDWMVQQNLNSEWLFPSTSRPDRPITEKQFYKIMARVGDLLSINYLGTHTMRKTGAYRVYTQSNYNIGLVIHLLNHSSEAMTLTYLGLD QASRETMLDQIDFG 112 Cluster: G1FE57MDQKEVSQNQTKYIQFRLSEEQYNKLKISGETYGLS UncharacterizedPNLYAKKLAQKSHLKKPYLEHDQAKSLLLELSKQGT proteinNLNQIAKKLNQFDRMDNQDKELIEALRYTYGVLAQA QKGYQELWQQLQK 113 Cluster: H2A9L4MATIAKISNGASAASALNYALGQDRPMHEKTEQWLQ MobilizationDHQLERPVELTNCRAVAVGGTNGIDPFIAKEQFDVV proteinRQLHNQTKESNQVMRITQSFALDELNPKVQKDWQKANDLGVELAENLYPNHQSAVYTHLDGKNHVLHNHIIV NKVNLETGKKLREQKGESVQRAREMNDRLASRENWHILEPPKERQTETEKELIAKNEYSYMDDLRERINKSLQDVSVSSYETFKERLSDNGVILSERGQTFSYAFLDANNKQRRARETRLGSDFGKETILHELENRARQNEFSAVEQREPAITPLERDTQQRESEIVSLEQAIEPRKSEALKRESKINRFIDTIKQFAGRVPELTQRVTRKLKQTKDKILDDFERRFSKDMKNYEQEQQKSLEKQANRDVQSEK KPTKDHDRGMSR 114 Cluster: S6EPU9MNKDEQLVVQVLNAYKNGKIDFSNVPELDRLVRQEV PutativeNKDFRDYQEKIEAVANQKIESAIQEQLHRLEAENLKA mobilizationTILKDIQVEKQALLALKKELNEQKEQIKADRKREIVER proteinYGILIANIVCLFCFLVVGILIGRWIYKGIWDGWGLHILYDTVMEIKPKHPYGAVILGLGGFGLIGAGIYGSFRLMY TASTWFDQRPKIFKRIFPKK 115Adenosine_ Q7DDR9 MVLDNKLGLTNSAELAKQEELLTKKRAKELFESGKIE monophosphate-DLEIGTFQGLSDIHQFLFQDIYDFAGKIREVNIAKGNF protein_QFAPRIFLAQTLEYIDKLPQETFDEIIDKYSDMNVAHP transferaseFREGNGRATRIWLDLILKNKLHKIVDWNQIDKDEYLNAMIRSTVSTNELKYLIQKALTDDLGKEQFFKGIDASYY YEGYYEIKTEDL 116 Cluster: O54680MSIITEFEKNQKQVKALNELSKRKVVEHNSLITSIAKM RepBDKTPLKMFELAVSCINTEAPPKDHTVYLSKTELFAFFKVSDNDKHSRFKQAVENMQKQAFFKIQEKKEYGFEFENIVPIPYVKWADYHDEVTIRFSPEIMPYLINLKQNFTQHALSDIAELNSKYSIILYRWLSMNYNQYEHYSAKGGRREEQVETYRNPSISIRELREMTDTMKDYPRFQSLESYIIKNSLKEINEHTSFKVTYEKVKKGRSINSIVFHITKKRRADDNSYKLEDKVYQKAKVQKEQKENLLYAEA MQSKYTKLLLEHFLLSPYEMTNPATMAGLQRNVYPKYDELKDLMGIDGVKKHLSYIYDKQEPYSKGNIAKYLK KAIEQYLPTVKRRGL 117 Cluster:G0WJS1 MSDNLKTIKELADELGVSKTAINKKVTDRERKLWFSK Replication-IGNKFVINEDGQKSIKRMFEGLTENQESQTENLEQKP associatedNSQTENFRNNNESNADIKYILDIIEYQKEQIKDLQNTK protein RepXDEQFKQLSNMQNLLDQQQRLALQDKKLLEEYKSEN DRLKVLKMPSQETKEEQANIQPQEELETLKEQTRALNDKIKGQEELNNKSSKKWYQFWK 118 Cluster: G0WJS2MFSYIYIILSYNTIKVKEVLKFEYRICTSFNWTSKFAEE TruncatedMKTCFFNSGFKFKNFKGLDNRNAKEKSELISEAEVVI peptidase ELAGGHVPTQNIFFQQINLKNMSPVRIF 119 Putative_O- P37746MQIAKNYLYNAIYQVFIIIVPLLTIPYLSRILGPSGIGINS antigen_YINSIVQYFVLFGSIGVGLYGNRQIAFVRDNQVKMSK transporterVFYEIFILRLFTICLAYFLFVAFLIINGQYHAYYLSQSIAIVAAAFDISWFFMGIENFKVTVLRNFIVKLLALFSIFLFVKSYNDLNIYILITVLSTLIGNLTFFPSLHRYLVKVNYRELRPIKHLKQSLVMFIPQIAVQIYWVLNKTMLGSLDSVTSSGFFDQSDKIVKLVLAIATATGTVMLPRVANAFAHREYSKIKEYMYAGFSFVSAISIPMMFGLIAITPKFVPLFFTSQFSDVIPVLMIESIAIIFIAWSNAIGNQYLLPTNQNKSYTVSVIIGAIVNLMLNIPLIIYLGTVGASIATVISEMSVTVYQLFIIHKQLNLHTLFSDLSKYLIAGLVMFLIVFKISLLTPTSWIFILLEITVGIIIYVVLLIFLKAEIINKLKFIMHK 120 Cluster: O50546MNLFGDSDYLEKLSSKGDPLERLEKVVDFECFRPTL TransposaseNRIFKYDLKNKSHGGRPPYDLVLMLKILILQRLYNLSDDAMEYQMIDRISFRRFLKIDDKVPDAKTIWNFRNQLSKSNRGNWLFSAFQEKLESQGMIAHKGQIVDATFIEA PKQRNPKDENELIKANRVPVNWTKNKRAQKDTAARWTIKGNERHYGYKNHIAIDTKSKFVKNYQTTPANVHDSQVIGVLVDPDEITLADSAYQNKATPKGAELFTFLKNTRSKSLKADDKMFNKIISKIRVRIEHVFGFVENSMHGSSLRSIGFDRAVLNTDLTNLTYNLLRHEQVKRLNLK TWR 121 Cluster: Q9RCJ9MRKYMIYLSSLLVTFILSYATITWLIMPVLTRYQSLARL Orf14.9INHFDYTALTLILLLTLIIWLFGIQYHLKHFSVIYLYLAFSVYLLLLFMVIFNKTTDFQAISLNPFDFIKADTRTIQEAVLNIIYFIPLGGLYCINTDFKQFVIISLVTLLGIETIQFIFYL GTFAISDIILNFLGCLIGYYCCWEIKKS122 hypothetical_ MDETYIKIKGRGHYLYRTIDADGLTLDIWLRKKRDTQ proteinAAYAFLKRLHKQFGEPKAIVTDKAPSLGSAFRKLQSVGLYTKTEHRTVKYLNNLIEQDHRPIKRRNKFYQSLRTASSTIKGMETLRGIYKNNRRNGTLFGFSVSTEIKVLM GITA 123 Putative_ P71057MKKNVLLSIIVPIYNVEKYIGSLVNSLVKQTNKNFEVIF glycosyltransferase_IDDGSTDESMQILKEIIAGSEQEFSLKLLQQVNQGLS EpsHSARNIGILNATGEYIFFLDSDDEIEINFVETILTSCYKYSQPDTLIFDYSSIDEFGNALDSNYGHGSIYRQKDLCTSEQILTALYKDEIPITAWSFVTKRSVIEKHNLLFSVGKKFEDNNFTPKVFYFSKNIGVISLRLYRYRKRSGSIMSNHPEKFFSDDAIFVTYDLLDFYDQYKIRELGAVVGKLVMTRLAFFPDSKKLYNELNPIIKKVFKDYISIEKRHTKRI KMYVKMYVFSSYVGYKLYRLVKGKHWK 124hypothetical_ MNHFKGKQFKKDVIIVAVGYYLRYNLSYREVQELLYD proteinRGINVCHTTIYRWVQEYSKVLYDLCKKKNRQSFYSWKMDETYIKIKGRWHYLYRAIDADGLTLDIWLQKKRDTQAAYAFLKRLHKQFGEPKAIVTDKAPSLGSAFRKLQSVGLYTKTEHRTVKYLNNLIEQDHWPIKRRNKFYQSLRTASSTIKGMETLRGIYKNNRRNGTLFGFSVSTEIKV LMGITA 125 Cluster: H5SYB4MQQNLLKYYGMTHSFSRRGYPYHNASLESWHGHL TransposaseKREWVYQFKYKNFEEAYQSIFWYIEAFYNSKRIHQSL GYLTPNQFEKVSA 126 Replication_P03856 MNDLEKRKVVEHNSLITSIAKMQKTALKMFELAVSCI initiation_DTENPPKDNIIYLSKKELFAFFDVSSASKHTRFKEAIE proteinLMQKQAFFQIKEVKDKGYEMTSIVPIPTVKWNSYNDDVMIQFNQFIMPYLIDLKAEFTQYKISELKELNSKYSIILYRWLSMNYNQYEHYNVKGGRRAEQVENYRKPSISVKELREITDTVNEYKEIYDFEKRVLKKSLAEINAHTSFNVNYEKIKKGRSIDSIVFHIEKKRMADDNSYKLGDKDYQDDKKQKSRNEADLLKQAMESKYTRLLSENFLIGMNDIMDTTTMVGLQKNVYPLYDELKELRGLNGVKDHLSYVSSKREEYSKHNIAKYLKKAIEQYLPTVKRQDLEN E 127 Cluster: A0A0D4CCQ1MNDNLKTIKEVADELGVSKKKIENKLSYIKKKGNTLG RepX-likeKVIGGVRYLNKQEIKILNISPETSKAPETSKVPETSKV proteinPETSKVPETSKVPETSKAPETSEVPETSKVPDKHVFSSSFDLLREQTAYLLKELEEKNKHIEKLIDNEKSMQNLLDQQQRLALQDKKLLEEYKSEINELKALKMPQEDMKDDSSIRGEAQEEIVRLKAQLKLSEEERNKAKEKEPV KTESKKWWQLWK 128 Cluster: G6FEV8MNFGEVLQTKRKSMGLTQEDLADKLFVSSKTISNWE UncharacterizedTNKTTPDIDNVIRISQLFDISLNNLLLEGSNMVENIKKK proteinAEINNLKKYSYCTVITDLVFLFIILSSHYGAELPISILIATCIGIGVNIAVMFYFLNRIKILEDKTKKQQRKEIFITIILCI LAFVVTILVSWFKH 129 Cluster:G6FEV7 MIDLEEEGFLVLWGISIASSYTETISTLQQSGGSAIFT UncharacterizedFLTYAIGLLFFILTVLPTNAVTTKSDNGFILFFLRAK protein 130 Cluster: A0A0B8R3X5MNYIKKFFIVLRLAILSQIGVAVYGGAKGFSLENGAHK ExosortaseLSLLAVLILIIFIVGNIYLLMYLGKKLGFLTLSKDFLTKK E/protease,NIIYILVGTLIARTAGIGGTLLLNATGVTQTANDETIGQ VPEID-LFTGENPLLIILLIGIAAPIMEEIVFRGGIVGYLFKDLPV CTERMVGIIVSSVLFGLMHSPTNIISFLIYGLIGLTCAIAYFKTR system, xrtERLEVSIAIHFLNNILPALVLAFGIS 131 Cluster: S6FVR0MKKIKNRERIIQKKFFVNEKEDERIKLMMRKTGITNFS MobilizationIFARRACCNKEIFSIDFSEYKNIISEISATKSELKRIGNN proteinINQIAKHLNENKNNQTKELMSDYQKQLENLEDKIQKV VHYISEG 132 Cluster: Q93T03MAKKQNYIWRNDRNFALDEYEQQQYYYVVESNDIIN ReplicationKARHDLTARELKLMDFVISKIQPEDTQFNVIKTSMYE proteinLTKVLNIKQNGKNYGDMAKAIGDLRKKEVLIYDDVHRTVTQTGWVQSAKYQENGQVEIKLNEDLAPHLLGLKTHYTQHLLIDTTKLKSRYSILLYKLMREADKDKGNSIAILQGTPEEFKEWLGAPKDYEYKDLKRNILKKAVEEINLKIDDMDLEILQGRCGRKVVQVEIHNNWTVQRAIEENS EYVESITTHDWLKGDSK 133 Cluster:G9ZK11 MIYTSGYFIAFLGLIIMLFNFKDLYPKLNIWCRLGFILL UncharacterizedCLGLILPMLFGFITGFINNH protein 134 Cluster: O53072MAREKSDIEYQVVTVRFPKEIYQEYKKILKSEGKIPTY UncharacterizedDLRNYIFSVVDEYEKGQR protein 135 hypothetical_MNYFKGKQFQKDVIIVAVGYYLRYNLSYREIQELLYD proteinRGINVCHTTIYRWVQEYSKVLYHLWKKKNRQSFYSWKMDETYIKIKGRWHYLYRAIDVDGLTLDIWLRKKRDTQAAYAFLKRLHKQFGQPRVIVKDKAPSIGSAFRKLQSNGLYTKTEHRTVKYLNNLIEQDHRPIKRRNKFYQSLRTASTTIKGMETIRGIYKKNRRNGTLFGFSVSTEIKVL MGILA 136 Cluster: A0A0E2UHK8MAGYNVLDDAKARNLGLDILEVKETEYAVVPVKGSV AraC familyPDSIHQAWKYLLEEFFPENGYKHSGLPDFEVYTENDI transcriptional HDPNYEMELWVPISKQregulator 137 Cluster: D2BRG5 MIIVAVGYYLRYNLSYREVQDLLYDRGINVCHTTIYRTransposase WVQEYGKLLYQNGFYQGTEHRTIKYLNNLIEQDHRP of IS1216E,VKRRNKFYRSLRTASPTIKGMEAIRGLYKKTRKEGTL IS6 family FGFSVCTEIKVLLGIPA 138Cluster: Q48724 MIKNHWMKKLKYLSLFFLLFAIYWFPDVILAYPEVYLK UPF0177SLVGYERQVVATWIFLGNMSISLFLGILICYKLGYYKNTIS protein inIFKIKNLLFLLITTIILFVIYFFSYTYYNSHFITPGIAKT abiGiQAAFSIQIVFPFVQFITIAICAPIFEEAAFRTTIYRFFKN 5′regionDKIAYIVSCVGFAWMHTGPNPILIVYLPMSIVLTSIYHRRRVLGESILVHGVFNALLPIVIPLLQVITGLYYL 139 Cluster: A0A0M2ZU19MKYFVTTLSPSKNMGTMNWQTMILSDYCVNDSYWE UncharacterizedKAKRELSEEVQWVTQSDLYKKIKWNHDSNDDIILSKP protein VSIILETVKSDFPHANVWVYQ 140Cluster: A0A0M2ZU05 MEIQTNFQIISDEELSEIVGGGYPNNQSMNDVLHWL Bacteriocin-NGHNDGNPKQLPKWMCGLG type signal sequence 141 Cluster: A0A0M2ZV61MTKYIYPNLKDNQKYLLKIIDGILTSNNISSEEKKLFLIA UncharacterizedKSNIEKGRNFDPQISELISSLQYLVHSDDVLVFFEEAR protein KIMQINPGTGGSPYGWSNFESK142 putative_ Q60048 MAKITLNFQKRLQQHSNHLVILSAILIVLGYLGKYGVNQIW cadmium-IWNSTMIIASIIGFIPVAIHAYQAIKVKQISIDLLVSIA transporting_VIGALFIGEYEESAIVTFLFAFGGFLEKKTLEKTRSSIK ATPaseELTNMAPRTALSADGEEMDIDEVEIGDKLLVKTGRQVPVDGRIYQGSGYVNEASITGESREIRKEAGTKVFAGSILENGTIYVEAEKVGEDTTFGKIIELVEEAQDTKSPAEKFIDRFAKYYTPAVLVIAAITWVFSHNLELAITILVLGCPGALVIGAPVSNVAGIGNGAKRGVLIKGGDVMNTFSHIDTLLFDKTGTLTKGNTEVVVVKNYGASKELIDAVASAENESDHPLATAVVRMIGKFNPIKFEKTDVVKGQGIIADNLLIGNEKMMVVNHITISPEQKQDITEITDSGASVVLVAADNRLQLIYGIADEIRSGVKESLEELRHEGISRMIMLTGDNETTAKAVAAQLGIDEVRANLMPEEKAEVVKSLKNSGKKIAFIGDGVNDSPSLALANIGIAMGSGTDTAIETSDIVLMRSSFDELVHAYGLSKRTVANMTQNIVIAIVVVLFLLASLILGGTGLVPSFVNMGTGMFVHEA SILIVIVNGMRLIRYREK 143 Cluster:A0A0D6E0F2 MQNNYTSKGKHLTESERLLIERWHNKEKVSNREIAY TransposaseRLGKAPQTIHNEIQRGTVQLKYKTKYSAKIAQESYKTLRTHSKRSTKLNAQLDDQISKAVKNKISLEVIHQELKGVVCLRTLYNWISSGILSVAYHELLYPQYRKPKKQRVTQPKHMLGQSIEERPESVDERSEYGHWEIDTVLLTKEKGECLLTLTERKTRLEIIRLIPNKTTHSVNQALRGIEFLALSVTSDNGREFAKLSEALDCPVYYCHAYASHERGTNENHNRMIRRHLPKGTKKTTKQVVAYIENWMNNYP RKMFNFKTPNQMLIESI 144 Cluster:P94884 MNHFKGKQFQQDVIIVAVGYYLRYNLSYREVQEILYD TransposaseRGINVSHTTIYRWVQEYGKLLYQIWKKKNKKSFYSWKMDETYIKIKGKWHYLYRAIDADGLTLDIWLRKKRDTQAAYAFLKRLVKQFDEPKVVVTDKAPSITSAFKKLKEYGFYQGTEHRTIKYLNNLIEQDHRPVKRRNKFYRSLRTASTTIKGMEAIRGLYKKTRKEGTLFGFSVCTEIKVL LGIPA 145 Alpha- Q8L208MNSRIFQHNTFTTLSIGFYKGTITLKEALTHGKVGIGT acetolactate_LDTANGEVTIIDGIAYHGDSENQVRLVEENETMPYVA decarboxylaseMVEHQPIVKFTDNSVSNSEDFLSALTKRFPTANTAYTIVMTGQFKEVTVSSKPANNTRPYDEIMADQPYFTKENISGTMLGVWAPKHLTDLFGIGFHLHFVSEDKTFTAHVQNFITENLAIELGKITQIEQEFPDEDENFDQHLFQ 146 DNA- P03013MNIGYARVSTGLQNLDLQKDSLKKYNCEKIFTDHMS invertase_GSKRERPGLKSAIEFSRPGDTIVVWRLDRLGRNMED hinLINIVNSLNNKGVSFHSLQENITMDKSSSTGQLMFHLFAAFAEFERNLILERSAAGREAARARGRLGGRPEKFSEQDVKLLKTLVESGTPIKSIADSWGVSRTTIYRYINK F 147 Cluster: D2BPF3MKIITATLLLVISLLGILGTAFLYLGELTQGKGGGFLFIL UncharacterizedGCFLILGIQSFTWLEILFGKRQNGEVKKYDYFLFNILK protein VIFSIGALQLFIQRCFF 148Nitrogen_ P29286 MSKYKHHFSHHEHHCVQLVPLFGLLSESELVQVEQV fixation_VNHKIFEKGETVISPFAVPQLAIVAHGTLKIYQLSSAG regulation_KEQLLRVIEPGGYAGEDALFGVMNDNLYGETLEETQ protein_ICFLRQQDFKNLLLKYPELSLKLLETTVRRAAEMQYQ FixKAQFLMMEDVESRIANYLLQLVKVVDSNSVMIPMKMKDLATFIGTTPETISRKFKILEEKGFIERRGKIIKILDID SLEDDYA 149 Cluster: Non-A0A161UM95 MMTKLMIDEKYAKELDKAEIDHHKPTAGAMLGHVLS specificNLFIENIRLTQAGIYAKSPVKCEYLREIAQKEVEYFFKI DNA-bindingSDLLLDENEIVPSTTEEFLKYHKFITEDPKAKYWTDE protein Dps/DLLESFIVDFQAQNMFITRAIKLANKEEKFALAAGVVE Iron-bindingLYGYNLQVIRNLAGDLGKSVADFHDEDEDNDN ferritin-like antioxidant protein/Ferroxidase 150 Cluster: A0A0M2ZU22 MSKVIMRLNELSCPSCMAKIEAAMTTTKGVANAKVLCopper ion FNASKVKAEFDENVVSADELISKVEKLGYPVLSSKVT binding VV protein 151Cluster: P94884 MNHFKGKQFQQDVIIVAVGYYLRYNLSYREVQEILYD TransposaseRGINVSHTTIYRWVQEYGKLLYQIWKKKNKKSFYSWKMDETYIKIKGKWHYLYRAIDADGLTLDIWLRKKRDTQAAYAFLKRLVKQFDEPKVVVTDKAPSITSAFKKLKEYGFYQGTEHRTIKYLNNLIEQDHRPVKRRNKFYRSLRTASTTIKGMEAIRGLYKKTRKEGTLFGFSVCTEIKVL LGIPA 152 hypothetical_MKMLRVQKPLLFKFSQIQVLQYTKTQDAVYKVNSNTI protein CSVYKLSFTLVQLRL 153Tyrosine_ MF_01808 MTYIELNPVNNVVLPKHNSSVEDFEISENKTITYDELK recombinase_IVLEYCHKHNKNQRLTLIIEFLFLTGLRLEELGGLQKS XerCSVDFKKQTIKIKHVIDTKAIGDNSRKLYLPKTFASRREIYVNDRCIEILKWFFDNSLDDDFVFTTMIGTTVKQSATYLFVRNVCEASLGKQKNRKYNVHMLRHAHISLLAELDIPIKATMKRVGHSQESTTLRIYSHVSQKMNDSIMRK LNEI 154 Cluster: P94884MNHFKGKQFQQDVIIVAVGYYLRYNLSYREVQEILYD TransposaseRGINVSHTTIYRWVQEYGKLLYQIWKKKNKKSFYSWKMDETYIKIKGKWHYLYRAIDADGLTLDIWLRKKRDTQAAYAFLKRLVKQFDEPKVVVTDKAPSITSAFKKLKEYGFYQGTEHRTIKYLNNLIEQDHRPVKRRNKFYRSLRTASTTIKGMEAIRGLYKKTRKEGTLFGFSVCTEIKVL LGIPA 155 Replication_ P13921MKQKKREQRSNKWAFLIYQESVPEDYLNLLEELHVP protein_FILSPWHDKDVNRTTGEFKKPHKHGVFFFESLKSYS RepBQVSELISDKLNSPEHVEVVMSPKGMYDYFTHAENPEKSPYNIEDIESGAGFELDKFLAENNEDLLNQVYEVMRDSGIKEFADFTDLIAKQFPDLLYFVFSKSYFFKIYLDS KRYIEIKQKDDEDNHGK 156hypothetical_ MENNYPYLLNREQASKFIGIRDDTFSVFFIVKIS protein 157 Na(+)/H(+)_P26235 MEDIFQITIILFFSMLATLLSKKLKIPEVVGQMLIGIILAP antiporterSVLGLINGGHTIEVMSEIGVILLMFLAGLESDLEVLKK NLKPSILVVLLQSLKIKRALSELQIS 158hypothetical_ MNHFKGKQFKKDVIIVAVGYYLRYNLSYREIQELLYD proteinRGINVCHTTIYRWVQEYSKVLYHLWKKKNRQSFYSWKMDETYIKIKGRWHYLYRAIDADGLTLDIWLRKKRDTQAAYAFLKRLHKQFGQPRVIVTDKAPSIGSAFRKLQSNGLYTKTEHRTVKYLNNLIEQDHRPIKRRNKFYRSLRTASTTIKGMETIRGIYKKNRRNGTLFGFSVSTEIKVL MGIPA 159 Cluster: I6TH45MSEHLNMASIKKKQPNRKERKQISFRVSEPEYLNLE MobCRSAKVLNISVPAFVKKKAQGARVVAPKINPDDSKEM mobilizationARQLAALGNNVNQLAKRVNQIEFADKDTQERLSADL protein RRTLHGLGEIWRQLT 160Cluster: A0A1V0PDY6 MATTHIKRSNGASRLVNYAEKRAVQKDGYNLDIEYA Relaxase/KSELKQVREIYGNKGATQAYASRVAFSPKEFDPKNV MobilizationKDQLKALEIAKEIYSTAYPNQQIAMYVHNDTDSLHVH nucleaseAVIGAINLLTGKKMHGNWQEYRERLVKITDKVVEKH domainGLTVTVPHPRPEKRTMAELKMKARGQVTWKDKIRQAVDTTMREAHISDFKSFKEKLGELAVNVIERGRDLTYTLTGTDYKSRGAKLGEDYKKETIFYELDRRNQLQYG TSRQRQGRAWLEGRGERLEQEQRARQNLAKRAEDLQRRTLESTEQSIQPSHQRPQKSKERGLGGPSL 161 Cluster: A0A1V0PDZ8MVHEIVQYHNDFNTVPLRGFNERERRIVMALLHQVK ReplicationNKDVEVVQLDFDTLRGLSGWNDTLAKSENSNAKFN initiatorRYLENLSDKIMTLRGTLRSEDGLQVVKFSLFPTFIIDG proteinKNTMTLKVQINPTFKYLTNIFDMFTAFELDDYNRMNTSYGQELYRLLKQYRTSGFYRVKIEDLRHLLSVPESYTNAKMDQKVFSKTTVTDLTNAFPNFKIKQERGTGRGRPIIGYTFTFDKEAPNKYELDRKKQEQIAQFWKSNDPEPMPNAVAQTEYQNPELRKEKEELEKHNASFGDLLK GWFKK 162 Cluster: A0A1V0PDX6MKFKKKNYTPQVDEKDCGCAALSMILKTYETEKSLA CAAX aminoSFLLNQRIKMHKVFEKIITIFFAFFLFFISQIPIYYVNYK terminalNKENNLYGISNKISLPFIFIALFVIIIAVALGKKRGFYHHSK proteaseKTLEFKNIMLILVLVTISIILNILINRFIIFHHLGIMNNQI family proteinNIDSILSSLSCLGKIFGIALLAPILEESIFRASIYQIFNNDKVSFLISSLLFAFLHSGYSWVFFTYLPVSLCMTFIYHR RKILTDSILFHSLFNLLVLGLNFLI 163Cluster: A0A1V0PES5 MLDILNKARIHKKWFLFSYSIISFCITIIYIVFNHTFFKVN PutativeWAKYNSDDSYKNKVDEILKHGVFWINGNLTSISSPLL transporterICLFLLGAFFSLTIFFLTWRNLSTRTWTPIISFLGFLIPFIHSDGNFINLLILSFILILFGAISSVPSLRYF 164 hypothetical_MNHFKGKQFKKDVIIVAVGYYLRYNLSYREIQELLYD proteinRGINVCHTTIYRWVQEYSKVLYHLWKKKNRQSFYSWKMDETYIKIKGRWHYLYRAIDADGLTLDIWLRKKRDTQAAYAFLKRLHKQFGQPRVIVTDKAPSIGSAFRKLQSNGLYTKTEHRTVKYLNNLIEQDHRPIKRRNKFYRSLRTASTTIKGMETIRGIYKKNRRNGTLFGFSVSTEIKVL MGIPA 165 Cluster: O54674MSIIPEKQNNQKQVLTLNELSKRKVVEHNSLITSIAKM RepBDKTPLKMFELAVSCIDTEKPLEDNTVYLSKRDLFAFFKVSDNDKHSRFKQAVEKMQKQAFFQIKEEAGKGFKFKSIVPIPYVEWTDYNDEVKIEFHREIMPYLINLKKNFTQHALSDIAELNSKYSLILYRWLSMNYNQYEHYSVK GGRRAEQVEAYRNPSIKVKEMRLMTDTVNEYHKYNDWDRYILKNSLKEINAHTSFNVTYDKIKKGRSIDSIVFHIEKKRMADDNSYKLGDKDYQEDKARKAETEDMLTLQALKSPYTKLLMEHFLLSYLDLTDTKILSGLQAHVYPLYDELKDLRGLNGVKDHLSYVRAKREDYSKKNITKYL KKAIEQYLPTVKRQDL 166 Cluster:Q2VHR8 MSEKLKTIKELADEIGVSKQAVWQKIKKESSIDLRQFT Replication-SKKGNTVYVDVDGQKVIKSAFF associated protein RepX 167 Cluster: G9BNK7MVKKLLRVLFFNKTSTKKRQQKVFVDDNVNNSVDG ReplicationNPEGNEEILFLRNLVSELQSEKKDLHKLLDQQQRLAL protein XQDKKLLEEYKAENDSLKALKMPTEGSQAEQANSQPKEEVKALKFEIRTLQEELNKQKIHSQEEREKLKAELTT PKKWYQFWK 168 Cluster: A0A0H1RR04MNKLKKLQELEAKSDKQAELMGELEARLGLIENKQI Uncharacterized protein 169Cluster: G8P721 MANTETVIWKSVKGFEGQYEVSNTGLVKSFKGKTER UncharacterizedIDRFDSNVQEILKRLSYDDCRRYKR protein  170 Cluster: G8P722MNMKNKTNENFVQIPNKMFMNINNDEKLVYVKLLQ UncharacterizedSQMIGYLDKDNRTTMTTVSLLVTLLGWSKGQYSNKK proteinVVKALNGLKDKKYINFESIQDVFTVQINKWNDKEEHIVPVDWKQSGVKFSGHTQIRLSVIDNLLEGKDFTLYA YTEYRKMKTHQYRICYEEWGFVLRMTKDGAFKNVNSSEVIIKVSNGFDSDTKRRETNSYLTFDSVEDVKEVSLKPTYKAQSSKSVVKEQEPELVEDDFDNFEEEELSFKAEAKKPLIKEKKITKKQANELKDEINKFFGNTMEDNIFKKMASDKRITSVEQAMEIQDINKPMSLEMWKVVQD SDNFFVRESGNKKLKNKAWQKKFWSDLKEEIDKAKELAYKTKFTSKYLYNTITEYYVDGGECVISSDKLYDY VHNRRVYSNDEYTYFTPTNMVPHLKFIKVTEKY171 Cluster: A0A0V8EN50 MYFCYSNKQKDFLNQKGIDSLFSARHAKTNKLFYVFUncharacterized YQSEELGQALTEFTEKKAEFFKNN protein 172 Carbon_ P15078MKDIGNSSNFTEDEELFLLRNKQGKIVGIKDLKQANF starvation_QETMKDWKKHLPKPSLLSIIIWVAVALLGGLAWSLIAL protein_AAQGETINAIWFVIAAVCSYLIGYRFYALYIQRKIMRPNDLRATPSESHNDGKEFDPTNRVVLFGHHFASIAGAGPLVGPVLAAQMGYLPGTIWIIFGVIFAGGVQDMLVLWYSHRRRAKSIGAMAHDEVGRFAGGLTSFIVFIMTMIVLAVLALICVTAMANSAWAVFSIGMTIPIALLMGIYLKYIRPGHVNEISAIGFILLLVAIFGGRWVSESSFAHIFMLSPTALVWWVMGYTFIAAIIPAWILLTPRDYLSMFMKIGTIAVLAIAVVGVRPDVTIPALTNFAHNTDGPAFAGSLFPFLFVTIACGALSGFHVMMSSGTTPHLIAKESQTRMIGYGGMLFESFVAIMALVAAISLNPGIYYSMNTPQASIQKLAASSYQADKSAEYNAAKAIPNVAMMPDGSKLSID WEGTTGEKALEQVAKDVGEQSIVSRTGGAPTLAVSMSNILHKVPLIGGTNMMGFWYHFAIMFEALFILSAVSAATKSTRYLLNDALRGFKKLGRLGDDDWLPSKIITTAVIVGVWGALLLMGVSDPNGGIKIMYPLFGISNQLIAAVALAIVCVMVIRKGYLKWVWIPALPLVWDVCVTFAASWQKIFSNDVNIGYFASYSAAKAQVASGKISGLALTNTQATMRNTMIQGSLSVIFLLCVAILLVICALKVAKILRTNEVGDKFSSEEVFEESNLFETSSFWPSKLEHKVLKSK VNE 173 hypothetical_MNYFKGKQFQKDVIIVAVGYYLRYNLSYREIQELLYD proteinRGINVCHTTIYRWVQEYSKVLYHLWKKKNRQSFYSWKMDETYIKIKGRWHYLYRAIDVDGLTLDIWLRKKRDTQAAYAFLKRLHKQFGQPRVIVKDKAPSIGSAFRKLQSNGLYTKTEHRTVKYLNNLIEQDHRPIKRRNKFYQSLRTASTTIKGMETIRGIYKKNRRNGTLFGFSVSTEIKVL MGILA 174 Cluster: G8PA25MKTLIHEDLRGKIIYLQEEIPFGOGRLIEQLRLPFLSQK PutativeLLTIPLIVDLKLAEFIRRQLYYCSPKWLKLQEKYYQRG competenceENLLNLTFERSFIAPLGLNLLEVFDDEIPLHKFTQIKQ protein/NINLYYENFLINFQQNSFKAVYPPRFYAIMKKQKKDM transcription NE factor 175Oligoendopeptidase_ P54124 MAKNRNEIPEKLTWDLTTIYKTDKEWEAELTRIKSELF,_plasmid SLVEETDPGHLLDSAESLLTITEKMLSISQQVEKLYVYASMKNDQDTREAKYQEYQSKATALYVKFGEVYAFY EPEFLKISKEVYNKWLGELQKLKNYDHMFERLFAKKAHILSQKEEKLLAAAGEIFESPSETFEIFDNADIKLPMVKNESDEMIQLTHGNYSSLMESKNRGVRKAAYKALYSNYEQYQHTYAKTLQTNVKVHNLNAQIRSYDSARQAALANNFVPEKVYDVLMEAIHQHLPLLHRYIELRKKILGITDLKMYDIYTPLSNLDYKFNYEDGVKKAEEVLAIFGKEYKGKVKAAFEQRWIDVEENIGKRSGAYSGGSYDT NAFMLLNWQETLDDLFTLVHETGHSMHSAFTRENQPYVYGNYPIFLAEIASTTNENILTETLLKESKDDKERFALLNHWLDSFRGTVFRQSQFAEFEQKIHEADAAGEVLTSEYLNSLYGEINEKYYNLAVKENPEIQYEWARIPHFYYNFYVFQYATGFAAATFLAEKVVHGSTEDRQKYLEYLKAGSSAYPLEVIAKAGVDMESTDYLDAAFELFEN RLSELEKLVEKGVHL 176 Cluster:P94881 MVETYKRTSNPMMNRPVVKAELVEWMRSSQTQITG ORF4 ELASLASVPVLTRLFPLVprotein 177 Cluster: Q48667 MQKRYSKEFKETLIAFYHSGOSVTQLSKEYDVAPATIInsertion YKWIDLYSKSNESSVSKADFLELKRQLAKVKEERDIL sequence KKVLTIFAEKKKIS981 178 Serine_ P0ADI0 MKIGYARVSTFEQKLESQIEVLKEAGAEEVFQEKFTGrecombinase_ TTVERPQFNLVFKKLKDGDTLIVTKLDRLARNTREVL PinREIVQSLFNRGIKVHILNIGLIDNTPTGQLIFTIFSAFAQFERDLIVIRTQEGKNFAKLHDPSFREGRPQKFTEEQIQFAYELKQQGMTYKMIERKTGISIATQKRRFIKAKNQ AIDKDY 179 Cluster: Q52233MKEYFQGDEFKDISKNGKDRKWKERKINNLNLAKIF ReplicationDSLDYPDSFIFNIKSCAEYLNFKRSSDGSLRLFQMYT proteinCKNKQCAICSWRRSMKYQVQISKIVEEAMIRKPKGRFLFLTLTVENVSGEGLNNELSLLSEAFNRLMKYKKVSKNILGFLRATEVTINESMDTYHPHIHVLLFISPTYFKNKNNYISQDEWTELWKKSAKLDYRPIVDVRSIKPKNEKTSDIRSAILETAKYPVKPMELNYDSAKVVDDLQKGLYRKRQIAFGGLFKQIKKELELDDIENGDLINIGDEENPISD GEIISVLWNHERQNYYVR 180 Cluster:T0VQK1 MTCSNLTIHLHAKNRSKLFGSKKYALQELEAESTAFV UncharacterizedVANHLNIDTKDYSIGYLNSWGFDKISDEQLENVIKND protein KLSNNKIKGENE (Fragment)181 Cluster: T0VLA4 MINYQGEDFTETEFYGREILEAIQLTNKFPTPKKVLIDUncharacterized MLEEMIHEQLDFIDKEELNNYINAKKYVQTLTEDEVK proteinNLCFEVKDLYEDVLKEFEIKL

TABLE 4 Selected Exopolysaccharide producingLactococcus lactis cremoris Proteins not foundin Lactococcus lactis cremoris Strain A SEQ ID NO. name uniprot_idProtein Sequence 182 Cluster: A0A218PFY7MERKKKKKENIWAIIVPILIIISLIGAWAYALRDSLIPND ExopolysaccharideYTKTNSSDQPTKTSVSNGYVEQKGVEAAVGSIALVD biosynthesisDAGVPEWVKVPSKVNLDKFTDLSTNNITIYRINNPEV protein EpsLLKTVTNRTDQRMKMSEVIAKYPNALIMNASAFDMQTGQVAGFQINNGKLIQDWSPGTTTQYAFVINKDGSCKIYDSSTPASTIIKNGGQQAYDFGTAIIRDGKIQPSDGSVDWKIHIFIANDKDNNLYAILSDTNAGYDNIMKSVSNLKLQNMLLLDSGGSSQLSVNGKTIVASQDDRAVPDYI VMK 183 Cluster: A0A0M2ZR43MVDAYLDNNLGDDLMIRYFASYFYQHKIYLVESREHI PolysaccharideRKTFYDIPNIYFYSEEDYKMNEYDFQLHVTIGGSMFIL pyruvylDDFKKLIRFRHRIKNSRKIKKRNIPSAIIGCNLGPFDKR transferaseNFGLKLAKFELKYKNLVTVRDKQSKELLLRGFKRKKI CsaB, csaBNIKLFPDIIFSKVLYKSIPKYGLGMTLSQVFRVTNVEF 184 putative_ P71059MKNKFSIIVPVYNGESHIKKCIDTLLKQTYNDFEIIIIND glycosyltransferase_GSTDDTKSVLTKFYAKNLKVKIVNTSNKGVSFARNLG EpsJINQSSGQYLLFVDSDDELSINALKYLSIMLNKKDRDLILFGFSLTGDNNRKNDTSILKSIANQNTDCKMNILKSILSTKNNILGYVWRAVYSLDFIKKNNIFFETHLKISEDYLFLLQSVEHSNNLFVITEEFYKYNLGETSMSNKFVPTLLNDMVWVNNWIESNILTVYPQFFVGFNCLVANTYIRYVQNAIRNKEENFMLKYREIKINKRKYNFQRSINQVIFHLDKFDFKSKIGVILFRIHLDIVYELLFNIKERKN 185 Cluster: Q3ZK44MTNLNRKKFFINFQSLVFFILIIIYGLTTKNVMGGSGIF EpsHSIDSILKYGILFICISVEGYIFLKNGNERRETSENYNNFKYYFIIITFLSLFASFKQVHFSFRTVQSFIFIFIPMLYSYLILNNWTFRQINFSMKIALFLSVIEYLFSIRMGFSQIISSLASINYNNTNASALESSTFALLSLGFAAYFGYYKKNFLCKIVSLLFVIMTFKRVITLSGCILVILGILKIKNLRVNRFFLIVSTITLVSFSLIYYYSIQPQNILEISEKIGFSIRDFSTNRTDRLAWLSMTDFKSYGLGSTTDFMYKLWGVDLEMDIVQLILEVGAFGVIVFIYFYLRFSKSNLYAFSFMALLLLNSILSSGMMSTFSWIIILIAMSTIMEYKEGM 186 Cluster: Q3ZK46MIFVTVGTHEQPFNRLIQKIDELVRDGQIKDDVFMQI EpsFGYSTYEPKYTKWASVIGYNDMTAYFNKADIVITHGGPSTYMQVLQHGKIPIVVPRQEKFGEHINDHQLRVSKQVIKKGYPLILCEDVSALKICIESSRIRTDEFIKSNNKN FISNFKKIIAFEE 187 Cluster:Q9X491 MKIALVGSSGGHLTHLYLLKKFWENEDRFWVTFDKT EpsEDAKSILKEERFYPCYYPTNRNVKNTIKNTILAFKILRKEKPDLIISSGAAVAVPFFWIGKLFGAKTVYIEIFDRIDKPTLTGKLVYPVTDKFIVQWEELKKVYPKAINLGGIF 188 putative_sugar_ P71062MEFFEDASSPESEEPKLVELKNFSYRELIIKRAIDILG transferase_GLAGSVLFLIAAALLYVPYKMSSKKDQGPMFYKQKR EpsLYGKNGKIFYILKFRTMIFNAEQYLELNPDVKAAYHANGNKLENDPRVTKIGSFIRRHSIDELPQFINVLKGDMALVGPRPILLFEAKEYGERLSYLLMCKPGITGYWTTHGRSKVLFPQRADLELYYLQYHSTKNDIKLLSLTIVQSIN GSDAY 189 Tyrosine- P96716MAKNKRSIDNNRYIITSVNPQSPISEQYRTIRTTIDFK protein_MADQGIKSFLVTSSEAAAGKSTVSANIAVAFAQQGK kinase_YwqDKVLLIDGDLRKPTVNITFKVQNRVGLTNILMHQSSIEDAIQGTRLSENLTIITSGPIPPNPSELLASSAMKNLIDSVSDFFDVVLIDTPPLSAVTDAQILSSYVGGVVLVVRAYETKKESLAKTKKMLEQVNANILGVVLHGVDSSDSPS YYYYGVE 190 putative_ P96715MQETQEQTIDLRGIFKIIRKRLSLILFSALIVTILGSIYTF capsular_FIASPVYTASTQLVVKLPNSDNSDAYAGQVSGNIQM polysaccharide_ANTINQVIVSPVILDKVQSNLNLSDDSFQKQVTAANQ biosynthesisTNSQVITLTVKYSNPYIAQKIADETAKIFSSDAAKLLNVTNVNILSKAKAQTTPISPKPKLYLAISVIAGLVLGLAIALLKELFDNKINKEEDIEALGLTVLGVTSLCSNE 191 Cluster: A9QSJ2MMKKGIFVITIVISIALIIGGFYSYNSRINNLSKADKGKE PolysaccharideVVKNSSEKNQIDLTYKKYYKNLPKSVQNKIDDISSKN biosynthesisKEVTLTCIWQSDSVISEQFQQNLQKYYGNKFWNIKNI proteinTYNGETSEQLLAEKVQNQVLATNPDVVLYEAPLFNDNQNIEATASWTSNEQLITNLASTGAEVIVQPSPPIYGGVVYPVQEEQFKQSLSTKYPYIDYWASYPDKNSDE MKGLFSDDGVYRTLNASGNKVWLDYITKYFTAN192 Cluster: O06027 MNNLFYHRLKELVESSGKSANQIERELGYPRNSLNN EpsRYKLGGEPSGTRLIGLSEYFNVSPKYLMGIIDEPNDSS AINLFKTLTQEEKKEMFIICQKWLFLEYQIEL193 Putative_O- P37746 MQIAKNYLYNAIYQVFIIIVPLLTIPYLSRILGPSGIGINSantigen_ YINSIVQYFVLFGSIGVGLYGNRQIAFVRDNQVKMSK transporterVFYEIFILRLFTICLAYFLFVAFLIINGQYHAYYLSQSIAIVAAAFDISWFFMGIENFKVTVLRNFIVKLLALFSIFLFVKSYNDLNIYILITVLSTLIGNLTFFPSLHRYLVKVNYRELRPIKHLKQSLVMFIPQIAVQIYWVLNKTMLGSLDSVTSSGFFDQSDKIVKLVLAIATATGTVMLPRVANAFAHREYSKIKEYMYAGFSFVSAISIPMMFGLIAITPKFVPLFFTSQFSDVIPVLMIESIAIIFIAWSNAIGNQYLLPTNQNKSYTVSVIIGAIVNLMLNIPLIIYLGTVGASIATVISEMSVTVYQLFIIHKQLNLHTLFSDLSKYLIAGLVMFLIVFKISLLTPTSWIFILLEITVGIIIYVVLLIFLKAEIINKLKFIMHK 194 Putative_ P71057MKKNVLLSIIVPIYNVEKYIGSLVNSLVKQTNKNFEVIF glycosyltransferase_ IDDGSTDESMQILKEIIAGSEQEFSLKLLQQVNQGLS EpsHSARNIGILNATGEYIFFLDSDDEIEINFVETILTSCYKYSQPDTLIFDYSSIDEFGNALDSNYGHGSIYRQKDLCTSEQILTALYKDEIPITAWSFVTKRSVIEKHNLLFSVGKKFEDNNFTPKVFYFSKNIGVISLRLYRYRKRSGSIMSNHPEKFFSDDAIFVTYDLLDFYDQYKIRELGAVVGKLVMTRLAFFPDSKKLYNELNPIIKKVFKDYISIEKRHTKRI KMYVKMYVFSSYVGYKLYRLVKGKHWK

TABLE 5 Select Proteins from 13 kb plasmid ofLactococcus lactis cremoris Strain A SEQ ID NO. name uniprot_idProtein Sequence 195 Foldase_ P0C2B5 MKKKMRLKVLLASTATALLLLSGCQSNQTDQTprotein_ VATYSGGKVTESSFYKELKQSPTTKTMLANMLI PrsAYRALNHAYGKSVSTKTVNDAYDSYKQQYGENF DAFLSQNGFSRSSFKESLRTNFLSEVALKKLKKVSESQLKAAWKTYQPKVTVQHILTSDEDTAKQVI SDLAAGKDFAMLAKTDSIDTATKDNGGKISFELNNKTLDATFKDAAYKLKNGDYTQTPVKVTDG YEVIKMINHPAKGTFTSSKKVLTASVYAKWSRDSSIMQRVISQVLKNQHVTIKDKDLADALDSYKK LATTN 196 PIII- P15292MQRKKKGLSFLLAGTVALGALAVLPVGEIQAK type_ AAISQQTKGSSLANTVTAATAKQAATDTTAATTproteinase NQAIATQLAAKGIDYNKLNKVQQQDIYVDVIVQMSAAPASENGTLRTDYSSTAEIQQETNKVIAAQ ASVKAAVEQVTQQTAGESYGYVVNGFSTKVRVVDIPKLKQIAGVKTVTLAKVYYPTDAKANSMA NVQAVWSNYKYKGEGTVVSVIDSGIDPTHKDMRLSDDKDVKLTKSDVEKFTDTVKHGRYFNSKV PYGFNYADNNDTITDDKVDEQHGMHVAGIIGANGTGDDPAKSVVGVAPEAQLLAMKVFTNSDTS ATTGSDTLVSAIEDSAKIGADVLNMSLGSDSGNQTLEDPEIAAVQNANESGTAAVISAGNSGTSGS ATEGVNKDYYGLQDNEMVGTPGTSRGATTVASAENTDVITQAVTITDGTGLQLGPETIQLSSNDFT GSFDQKKFYVVKDASGNLSKGKVADYTADAKGKIAIVKRGELTFDDKQKYAQAAGAAGLIIVNN DGTATPVTSMALTTTFPTFGLSSVTGQKLVDWVTAHPDDSLGVKIALTLVPNQKYTEDKMSDFTSY GPVSNLSFKPDITAPGGNIWSTQNNNGYTNMSGTSMASPFIAGSQALLKQALNNKNNPFYAYYKQL KGTALTDFLKTVEMNTAQPINDINYNNVIVSPRRQGAGLVDVKAAIDALEKNPSTVVAENGYPAV ELKDFTSTDKTFKLTFTNRTTHELTYQMDSNTDTNAVYTSATDPNSGVLYDKKIDGAAIKAGSNIT VPAGKTAQIEFTLSLPKSFDQQQFVEGFLNFKGSDGSRLNLPYMGFFGDWNDGKIVDSLNGITYSPA GGNFGTVPLLTNKNTGTQYYGGMVTDADGNQTVDDQAIAFSSDKNALYNDISMKYYLLRNISNV QVDILDGQGNKVTTLSSSTNLTKTYYNAHSQQYIYYHAPAWDGTYYDQRDGNIKTADDGSYTYRIS GVPEGGDKRQVFDVPFKLDSKAPTVRHVALSAKTKNGKTQYYLTAEVKDDLSGLDATKSVKTAI NEVTNLDATFTDAGTTADGYTKIETPLSDEQAQALGNGDNSAELYLTDNASNATDQDASVQKPGS TSFDLIVNGSGIPDKISSTTTGYEANTQGGGTYTFSGTYPAAVDGTYTDAQGKKHDLNTTYDAATNS FTASMPVTNADYAAQVDLYADKAHTQLLKHFDTKVRLTAPTFTDLKFNNGSDQTSEATIKVTGTVS ADTKTVNVGDTVAALDAQHHFSVDVPVNYGDNTIKVIATDEDGNTTTEQKTITSSYDPDMLKNPV TFDQGVTFGSNEFNATSAKFYDPKTGIATITGKVKHPTTTLQVDGKQIPIKDDLTFSFTLDLGTLGQK PFGVVVGDTTQNKTFQEALTFILDAVAPTLSLDSSTDAPVYTNDPNFQITGTATDNAQYLSLSINGSS VASQYADININSGKPGHMAIDQPVKLLEGKNVLTVAVTDSEDNTTTKNITVYYEPKKTLAAPTVTP STTEPAQTVTLTANAAATGETVQYSADGGKTYQDVPAAGVTITANGTFKFKSTDLYGNESPAVDY VVTNIKADDPAQLQAAKQALTNLIASAKTLSASGKYDDATTTALAAATQKAQTALDQTNASVDSL TGANRDLQTAINQLAAKLPADKKTSLLNQLQSVKDALGTDLGNQTDPSTGKTFTAALDDLVAQAQ AGTQTDDQLQATLAKILDEVLAKLAEGIKAATPAEVGNAKDAATGKTWYADIADTLTSGQASADA SDKLAHLQALQSLKTKVAAAVEADKTVGKGDDTTGTSDKGSGQGTPAPATGDTGKDKGDEGSQ PSSGGNIPTNPATTTSTSADDTTDRNGQHTTGTSDKGGGQGTPAPATGDTGKDKGDEGSQPSSGGN IPTNPATTTSTSADDTTDRNGQHTTGTSDKGGGQGTPAPATGDTGKDKGDEGSQPSSGGNIPTNPA TTTSTSTDDTTDRNGQHTTGKGALPKTGETTERPAFGFLGVIVVILMGVLGLKRKQREE 197 Cluster: T0V9Y4MRAAEGLFVYNKTNFHYLPQNIAFADFKSGKFA UncharacterizedTSGMSMILIDSVNHRILDVMKDRGAGQLRAYFN proteinQYSPSARAAVKTITVDLFTPYRAMIKDLFPNANI VADRFHVVTQAYRELNKVRISVMKQFGSDSKEYRQLKRFWKLLMKHENALDYMTSKNRINFKHA YLTDKEVIDRLLALSDELRDAYAFYQVIL 198Cluster: T0UTW8 MDNDIRILIGLTDLNIDFDAKAEQHFNETNLNGT UncharacterizedAPITWNLLLTYATNCEKFGTPMVHNGIKMVTH protein KGPRIAFKFQNYRIRKQKFL 199Cluster: T0UZT2 MIENTINIAYARKFYKTKDYHSFCNLIKGNKGLF UncharacterizedGNKTVNQKANISFVKSEGEKHTHIYLDYQETCK proteinVAHPNFLQLINLLKNYDPEFSEEKLPTFDLNDKI FGEYEIKVIPISKTKIVNTIDDVMNEIAKEIVLKYNQDMFKVTSKLGEISLTPIQEKFDKLKDI 200 Cluster: RepB Q9AIQ4MIIPEKQNKQKQVLTLNELEKRKVVEHNALIQS VAKMQKTALKMFELAVSCIDTEEPPKNNTVYLSKSELFKFFEVSSSSKHSQFKEAVNYMQKQAFFNI KADKKLGIEYESIVPIPYVKWNDYNDEVTIRFDQAIMPYLIDLKAEFTQYKISELQKLNSKYSIILYRW LSMNYNQYEHYSVKGGRRADQVEAYRTPSIKVKELREITDTINEHQHFPHFETRVLKKAIEEINAHT SFNVTYEKVKKGRSIDSIVFHIEKKRMADDNSYKLEDKVYQEDKARKAETEKDLVFQAMQSPYTR LLIENMFLNVYETTDSQIMAGLQKNVYPLYDELKELRGLNGVKDHLSYVSSKQEAYSKRNVAKYL KKAIEQYLPTVKRQDLNHE 201 Cluster: Q7BLH6MSEDLKTIKELADELGVSKSYVDKIIRILKLHTK UncharacterizedLDKVGNKYVISKKQEKSIITRIENSKSTTETHTES proteinTTQSHTKVDAEVDFLKEEIAYLKSNHDKQLTNK DKQIETLSNLLDQQQRLALQDKKWLEEYKAEINDLKALKMPSEDTKEEQSNYRSLEKEKDFVQTIQ ESYESEIKVLNQKLAEQEEQIQEIQKEKETKEKKWFQFWK 202 Cluster: RepC O05547 MAQTFDRKILRALQDNGVREIRAYEVVSKRLTIFQTDRGTFKYSDSLYRLVAPRQELWRNCTTGFI SEEKYHFYKK 203 Cluster: Q2VHJ0MNHFKGKQFKKDVIIVAVGYYLRYNLSYREIQE TransposaseLLYDRGINVCHTTIYRWVQEYSKVLYHLWKKK NRQSFYSWKMDETYIKIKGRWHYLYRAIDADGLTLDIWLRKKRDTQAAYAFLKRLHKQFGQPRVI VTDKAPSIGSAFRKLQSNGLYTKTEHRTVKYLNNLIEQDHRPIKRRNKFYRSLRTASTTIKGMETIR GIYKKNRRNGTLFGFSVSTEIKVLMGILA

TABLE 6 Select Proteins from 30 kb plasmid ofLactococcus lactis cremoris Strain B SEQ ID NO. name uniprot_idProtein Sequence 204 Cluster: A0A218PFY7MERKKKKKENIWAIIVPILIIISLIGAWAYALRD ExopolysaccharideSLIPNDYTKTNSSDQPTKTSVSNGYVEQKGVEA biosynthesisAVGSIALVDDAGVPEWVKVPSKVNLDKFTDLS protein EpsLTNNITIYRINNPEVLKTVTNRTDQRMKMSEVIA KYPNALIMNASAFDMQTGQVAGFQINNGKLIQDWSPGTTTQYAFVINKDGSCKIYDSSTPASTIIK NGGQQAYDFGTAIIRDGKIQPSDGSVDWKIHIFIANDKDNNLYAILSDTNAGYDNIMKSVSNLKLQ NMLLLDSGGSSQLSVNGKTIVASQDDRAVPDY IVMK205 Cluster: A4VC87 MAQTIQTLALNVRLSCQLLDVPESSYYERINRH Transposase BPSKTQLRRQYLSLKISQLFNANRGIYGAPKIHHL of IS981LLKQGEKVGLKLVQKLMKQLQLKSVVIKKFKP GYSLSDHINRKNLIQTEPTKKNKVWSTDITYIPTQQGWAYLSTIMDRYTKKVIAWDLGKRMTVEL VQRTLNKAIKSQDYPEAVILHSDQGSQYTSLEYEELLKYYGMTHSFSRRGYPYHNASLESWHGHL KREWVYQFKYKNFEEAYQSIFWYIEAFYNSKRIHQSLGYLTPNQFEKVSA 206 Cluster: A0A0M2ZR43MVDAYLDNNLGDDLMIRYFASYFYQHKIYLVE PolysaccharideSREHIRKTFYDIPNIYFYSEEDYKMNEYDFQLH pyruvylVTIGGSMFILDDFKKLIRFRHRIKNSRKIKKRNIP transferaseSAIIGCNLGPFDKRNFGLKLAKFELKYKNLVTV CsaB, csaBRDKQSKELLLRGFKRKKINIKLFPDIIFSKVLYK SIPKYGLGMTLSQVFRVTNVEF 207 putative_P71059 MKNKFSIIVPVYNGESHIKKCIDTLLKQTYNDF glycosyltransferase_EIIIINDGSTDDTKSVLTKFYAKNLKVKIVNTSN EpsJKGVSFARNLGINQSSGQYLLFVDSDDELSINAL KYLSIMLNKKDRDLILFGFSLTGDNNRKNDTSILKSIANQNTDCKMNILKSILSTKNNILGYVWRA VYSLDFIKKNNIFFETHLKISEDYLFLLQSVEHSNNLFVITEEFYKYNLGETSMSNKFVPTLLNDM VWVNNWIESNILTVYPQFFVGFNCLVANTYIRYVQNAIRNKEENFMLKYREIKINKRKYNFQRSI NQVIFHLDKFDFKSKIGVILFRIHLDIVYELLFNIKERKN 208 Cluster: EpsH Q3ZK44 MTNLNRKKFFINFQSLVFFILIIIYGLTTKNVMGGSGIFSIDSILKYGILFICISVEGYIFLKNGNERRE TSENYNNFKYYFIIITFLSLFASFKQVHFSFRTVQSFIFIFIPMLYSYLILNNWTFRQINFSMKIALFLS VIEYLFSIRMGFSQIISSLASINYNNTNASALESSTFALLSLGFAAYFGYYKKNFLCKIVSLLFVIMT FKRVITLSGCILVILGILKIKNLRVNRFFLIVSTITLVSFSLIYYYSIQPQNILEISEKIGFSIRDFSTNRT DRLAWLSMTDFKSYGLGSTTDFMYKLWGVDLEMDIVQLILEVGAFGVIVFIYFYLRFSKSNLYAF SFMALLLLNSILSSGMMSTFSWIIILIAMSTIMEYKEGM 209 Cluster: A0A0M2ZW08 MKKLKISVIIRTYNEVKHIGEVLKSLTDQTYLNTransferase 2, HEIIIVDSGSVDGTLDIIERYPVKLVSINKEDFNY rSAM/selenodoSYASNVGVQNSSGDIVCFLSGHSVPVYKNYLE main-associatedKINEIFQETEIGACYGEVIALPDGSITEKIFNRIG YLKSKLSLNNKRFFLENKIHPGIFSCSNACARKKLLLKYPFKVELGHGGEDVEVAYRIIQDGYFV AKSVELLVMHSHGSSLKKFIKEYKAWGKMYEDVLKFIKKNNDKSQ 210 Cluster: EpsF Q3ZK46MIFVTVGTHEQPFNRLIQKIDELVRDGQIKDDV FMQIGYSTYEPKYTKWASVIGYNDMTAYFNKADIVITHGGPSTYMQVLQHGKIPIVVPRQEKFG EHINDHQLRVSKQVIKKGYPLILCEDVSALKICIESSRIRTDEFIKSNNKNFISNFKKIIAFEE 211 Cluster: EpsE Q9X491MKIALVGSSGGHLTHLYLLKKFWENEDRFWV TFDKTDAKSILKEERFYPCYYPTNRNVKNTIKNTILAFKILRKEKPDLIISSGAAVAVPFFWIGKLFG AKTVYIEIFDRIDKPTLTGKLVYPVTDKFIVQWEELKKVYPKAINLGGIF 212 putative_sugar_ P71062MEFFEDASSPESEEPKLVELKNFSYRELIIKRAID transferase_EpsLILGGLAGSVLFLIAAALLYVPYKMSSKKDQGP MFYKQKRYGKNGKIFYILKFRTMIFNAEQYLELNPDVKAAYHANGNKLENDPRVTKIGSFIRRHSI DELPQFINVLKGDMALVGPRPILLFEAKEYGERLSYLLMCKPGITGYWTTHGRSKVLFPQRADLE LYYLQYHSTKNDIKLLSLTIVQSINGSDAY 213Tyrosine- P96717 MIDIHCHILPGIDDGAKTSGDTLTMLKSAIDEGI protein_TTITATPHHNPQFNNESPLILKKVKEVQNIIDEH phosphatase_YwqEQLPIEVLPGQEVRIYGDLLKEFSEGKLLTAAGTS SYILIEFPSNHVPAYAKELFYNIKLEGLQPILVHPERNSGIIENPDILFDFIEQGVLSQITASSVTGHFG KKIQKLSFKMIENHLTHFVASDAHNVTSRAFKMKEAFEIIEDSYGSDVSRMFQNNAESVILNESF YQEKPTKIKTKKLLGLF 214 Tyrosine- P96716MAKNKRSIDNNRYIITSVNPQSPISEQYRTIRTTI protein_kinase_DFKMADQGIKSFLVTSSEAAAGKSTVSANIAV YwqD AFAQQGKKVLLIDGDLRKPTVNITFKVQNRVGLTNILMHQSSIEDAIQGTRLSENLTIITSGPIPPNP SELLASSAMKNLIDSVSDFFDVVLIDTPPLSAVTDAQILSSYVGGVVLVVRAYETKKESLAKTKKM LEQVNANILGVVLHGVDSSDSPSYYYYGVE 215putative_ P96715 MQETQEQTIDLRGIFKIIRKRLSLILFSALIVTILG capsular_SIYTFFIASPVYTASTQLVVKLPNSDNSDAYAG polysaccharide_QVSGNIQMANTINQVIVSPVILDKVQSNLNLSD biosynthesisDSFQKQVTAANQTNSQVITLTVKYSNPYIAQKI ADETAKIFSSDAAKLLNVTNVNILSKAKAQTTPISPKPKLYLAISVIAGLVLGLAIALLKELFDNKIN KEEDIEALGLTVLGVTSLCSNE 216 Cluster:A9QSJ2 MMKKGIFVITIVISIALIIGGFYSYNSRINNLSKA PolysaccharideDKGKEVVKNSSEKNQIDLTYKKYYKNLPKSVQ biosynthesisNKIDDISSKNKEVTLTCIWQSDSVISEQFQQNLQ proteinKYYGNKFWNIKNITYNGETSEQLLAEKVQNQV LATNPDVVLYEAPLFNDNQNIEATASWTSNEQLITNLASTGAEVIVQPSPPIYGGVVYPVQEEQFK QSLSTKYPYIDYWASYPDKNSDEMKGLFSDDGVYRTLNASGNKVWLDYITKYFTAN 217 Cluster: EpsR O06027MNNLFYHRLKELVESSGKSANQIERELGYPRNS LNNYKLGGEPSGTRLIGLSEYFNVSPKYLMGIIDEPNDSSAINLFKTLTQEEKKEMFIICQKWLFLE YQIEL 218 Cluster: Q2VHJ5MSVSIIDSFPIPLCQPIRNFRSKGLGDYANVGYN Transposase AATKGQYFYGCKCHALVSESGYVIDYTITPASM ADSSMTEEVLSQFGTPTVLGDMGYLGQSLHDRLELKGIDLMTPVRKNMKQKKILFPNFSKRRKVI ERVFSFLTNLGAERCKSRSPQGFQLKLEMILLAYSLLLKSAKSLEPETLRYSIGYQVMAK 219 Cluster: Signal A0A0B8QXZ2MTIKNKKDLSSSIEQLEKAINQQETILKKFDNEQ transduction LDFEQIKKLENLLIQEREKAKQVQIKINRSVLQN histidine kinaseNSENYKERKKRTRQLIQKGALLEKYLEAKHLT VDETEQLLQIFANMINKPELLVNFIGK 220Cluster: B1N0G0 MVQQIVLPIKDSNILKMVQDTLLDSFRAGRRN TyrosineYTIFQVGKATLLRVSDVMKLKKTDVFNSDGTV recombinaseKQTAFIHDQKTGKANTLYLKPVQQDLVVYHD WMVQQNLNSEWLFPSTSRPDRPITEKQFYKIMARVGDLLSINYLGTHTMRKTGAYRVYTQSNY NIGLVIHLLNHSSEAMTLTYLGLDQASRETMLD QIDFG221 Cluster: G1FE57 MDQKEVSQNQTKYIQFRLSEEQYNKLKISGET UncharacterizedYGLSPNLYAKKLAQKSHLKKPYLEHDQAKSLL proteinLELSKQGTNLNQIAKKLNQFDRMDNQDKELIE ALRYTYGVLAQAQKGYQELWQQLQK 222 Cluster:H2A9L4 MATIAKISNGASAASALNYALGQDRPMHEKTE MobilizationQWLQDHQLERPVELTNCRAVAVGGTNGIDPFI proteinAKEQFDVVRQLHNQTKESNQVMRITQSFALDE LNPKVQKDWQKANDLGVELAENLYPNHQSAVYTHLDGKNHVLHNHIIVNKVNLETGKKLREQK GESVQRAREMNDRLASRENWHILEPPKERQTETEKELIAKNEYSYMDDLRERINKSLQDVSVSSY ETFKERLSDNGVILSERGQTFSYAFLDANNKQRRARETRLGSDFGKETILHELENRARQNEFSAVE QREPAITPLERDTQQRESEIVSLEQAIEPRKSEALKRESKINRFIDTIKQFAGRVPELTQRVTRKLKQT KDKILDDFERRFSKDMKNYEQEQQKSLEKQANRDVQSEKKPTKDHDRGMSR 223 Cluster: S6EPU9 MNKDEQLVVQVLNAYKNGKIDFSNVPELDRLPutative VRQEVNKDFRDYQEKIEAVANQKIESAIQEQLH mobilizationRLEAENLKATILKDIQVEKQALLALKKELNEQK proteinEQIKADRKREIVERYGILIANIVCLFCFLVVGILI GRWIYKGIWDGWGLHILYDTVMEIKPKHPYGAVILGLGGFGLIGAGIYGSFRLMYTASTWFDQRP KIFKRIFPKK 224 Adenosine_ Q7DDR9MVLDNKLGLTNSAELAKQEELLTKKRAKELFE monophosphate-SGKIEDLEIGTFQGLSDIHQFLFQDIYDFAGKIRE protein_VNIAKGNFQFAPRIFLAQTLEYIDKLPQETFDEII transferaseDKYSDMNVAHPFREGNGRATRIWLDLILKNKL HKIVDWNQIDKDEYLNAMIRSTVSTNELKYLIQKALTDDLGKEQFFKGIDASYYYEGYYEIKTEDL 225 Cluster: RepB O54680MSIITEFEKNQKQVKALNELSKRKVVEHNSLIT SIAKMDKTPLKMFELAVSCINTEAPPKDHTVYLSKTELFAFFKVSDNDKHSRFKQAVENMQKQAF FKIQEKKEYGFEFENIVPIPYVKWADYHDEVTIRFSPEIMPYLINLKQNFTQHALSDIAELNSKYSII LYRWLSMNYNQYEHYSAKGGRREEQVETYRNPSISIRELREMTDTMKDYPRFQSLESYIIKNSLKE INEHTSFKVTYEKVKKGRSINSIVFHITKKRRADDNSYKLEDKVYQKAKVQKEQKENLLYAEAM QSKYTKLLLEHFLLSPYEMTNPATMAGLQRNVYPKYDELKDLMGIDGVKKHLSYIYDKQEPYSK GNIAKYLKKAIEQYLPTVKRRGL 226 Cluster:G0WJS1 MSDNLKTIKELADELGVSKTAINKKVTDRERK Replication-LWFSKIGNKFVINEDGQKSIKRMFEGLTENQES associatedQTENLEQKPNSQTENFRNNNESNADIKYILDIIE protein RepXYQKEQIKDLQNTKDEQFKQLSNMQNLLDQQQ RLALQDKKLLEEYKSENDRLKVLKMPSQETKEEQANIQPQEELETLKEQTRALNDKIKGQEELNN KSSKKWYQFWK 227 Cluster: G0WJS2MFSYIYIILSYNTIKVKEVLKFEYRICTSFNWTS TruncatedKFAEEMKTCFFNSGFKFKNFKGLDNRNAKEKS peptidase EELISEAEVVILAGGHVPTQNIFFQQINLKNMSPV RIF 228 Putative_O- P37746MQIAKNYLYNAIYQVFIIIVPLLTIPYLSRILGPS antigen_GIGINSYTNSIVQYFVLFGSIGVGLYGNRQIAFV transporterRDNQVKMSKVFYEIFILRLFTICLAYFLFVAFLII NGQYHAYYLSQSIAIVAAAFDISWFFMGIENFKVTVLRNFIVKLLALFSIFLFVKSYNDLNIYILITV LSTLIGNLTFFPSLHRYLVKVNYRELRPIKHLKQSLVMFIPQIAVQIYWVLNKTMLGSLDSVTSSGF FDQSDKIVKLVLAIATATGTVMLPRVANAFAHREYSKIKEYMYAGFSFVSAISIPMMFGLIAITPK FVPLFFTSQFSDVIPVLMIESIAIIFIAWSNAIGNQYLLPTNQNKSYTVSVIIGAIVNLMLNIPLIIYLGT VGASIATVISEMSVTVYQLFIIHKQLNLHTLFSDLSKYLIAGLVMFLIVFKISLLTPTSWIFILLEITVG IIIYVVLLIFLKAEIINKLKFIMHK 229Cluster: O50546 MNLFGDSDYLEKLSSKGDPLERLEKVVDFECF TransposaseRPTLNRIFKYDLKNKSHGGRPPYDLVLMLKILI LQRLYNLSDDAMEYQMIDRISFRRFLKIDDKVPDAKTIWNFRNQLSKSNRGNWLFSAFQEKLESQ GMIAHKGQIVDATFIEAPKQRNPKDENELIKANRVPVNWTKNKRAQKDTAARWTIKGNERHYG YKNHIAIDTKSKFVKNYQTTPANVHDSQVIGVLVDPDEITLADSAYQNKATPKGAELFTFLKNTRS KSLKADDKMFNKIISKIRVRIEHVFGFVENSMHGSSLRSIGFDRAVLNTDLTNLTYNLLRHEQVKR LNLKTWR 230 Cluster: Orf14.9 Q9RCJ9MRKYMIYLSSLLVTFILSYATITWLIMPVLTRY QSLARLINHFDYTALTLILLLTLIIWLFGIQYHLKHFSVIYLYLAFSVYLLLLFMVIFNKTTDFQAISL NPFDFIKADTRTIQEAVLNIIYFIPLGGLYCINTDFKQFVIISLVTLLGIETIQFIFYLGTFAISDIILNFL GCLIGYYCCWEIKKS 231 Cluster:Q2VHJ0 MDETYIKIKGRGHYLYRTIDADGLTLDIWLRK TransposaseKRDTQAAYAFLKRLHKQFGEPKAIVTDKAPSL GSAFRKLQSVGLYTKTEHRTVKYLNNLIEQDHRPIKRRNKFYQSLRTASSTIKGMETLRGIYKNN RRNGTLFGFSVSTEIKVLMGITA 232 Putative_P71057 MKKNVLLSIIVPIYNVEKYIGSLVNSLVKQTNK glycosyltransferase_NFEVIFIDDGSTDESMQILKEIIAGSEQEFSLKLL EpsHQQVNQGLSSARNIGILNATGEYIFFLDSDDEIEI NFVETILTSCYKYSQPDTLIFDYSSIDEFGNALDSNYGHGSIYRQKDLCTSEQILTALYKDEIPITAW SFVTKRSVIEKHNLLFSVGKKFEDNNFTPKVFYFSKNIGVISLRLYRYRKRSGSIMSNHPEKFFSDD AIFVTYDLLDFYDQYKIRELGAVVGKLVMTRLAFFPDSKKLYNELNPIIKKVFKDYISIEKRHTKRI KMYVKMYVFSSYVGYKLYRLVKGKHWK 233Cluster: Q2VHJ0 MNHFKGKQFKKDVIIVAVGYYLRYNLSYREVQ TransposaseELLYDRGINVCHTTIYRWVQEYSKVLYDLCKK KNRQSFYSWKMDETYIKIKGRWHYLYRAIDADGLTLDIWLQKKRDTQAAYAFLKRLHKQFGEPK AIVTDKAPSLGSAFRKLQSVGLYTKTEHRTVKYLNNLIEQDHWPIKRRNKFYQSLRTASSTIKGM ETLRGIYKNNRRNGTLFGFSVSTEIKVLMGITA 234Cluster: H5SYB4 MQQNLLKYYGMTHSFSRRGYPYHNASLESWH TransposaseGHLKREWVYQFKYKNFEEAYQSIFWYIEAFYN SKRIHQSLGYLTPNQFEKVSA

Production of Immune Modulating Lactococcus Strain EVs

In certain aspects, the immune modulating Lactococcus strain EVsdescribed herein can be prepared using any method known in the art.

In some embodiments, the immune modulating Lactococcus strain EVs areprepared without an EV purification step. For example, in someembodiments, immune modulating Lactococcus strain bacteria comprisingthe EVs described herein are killed using a method that leaves theimmune modulating Lactococcus strain bacterial EVs intact and theresulting bacterial components, including the EVs, are used in themethods and compositions described herein. In some embodiments, theimmune modulating Lactococcus strain bacteria are killed using anantibiotic (e.g., using an antibiotic described herein). In someembodiments, the immune modulating Lactococcus strain bacteria arekilled using UV irradiation.

In some embodiments, the EVs described herein are purified from one ormore other bacterial components. Methods for purifying EVs from bacteriaare known in the art. In some embodiments EVs are prepared frombacterial cultures using methods described in S. Bin Park, et al. PLoSONE. 6(3):e17629 (2011) or G. Norheim, et al. PLoS ONE. 10(9): e0134353(2015), each of which is hereby incorporated by reference in itsentirety. In some embodiments, the bacteria are cultured to high opticaldensity and then centrifuged to pellet bacteria (e.g., at 10,000×g for30 min at 4° C.). In some embodiments, the culture supernatants are thenpassed through filter to exclude intact bacterial cells (e.g., a 0.22 μmfilter). In some embodiments, filtered supernatants are centrifuged topellet bacterial EVs (e.g., at 100,000-150,000×g for 1-3 hours at 4°C.). In some embodiments, the EVs are further purified by resuspendingthe resulting EV pellets (e.g., in PBS), and applying the resuspendedEVs to sucrose gradient (e.g., a 30-60% discontinuous sucrose gradient),followed by centrifugation (e.g., at 200,000×g for 20 hours at 4° C.).EV bands can be collected, washed with (e.g., with PBS), and centrifugedto pellet the EVs (e.g., at 150,000×g for 3 hours at 4° C.). Thepurified EVs can be stored, for example, at −80° C. until use. In someembodiments, the EVs are further purified by treatment with DNase and/orproteinase K.

For example, in some embodiments, cultures of immune modulatingLactococcus strain bacteria disclosed herein can be centrifuged at11,000×g for 20-40 min at 4° C. to pellet bacteria. Culture supernatantsmay be passed through a 0.22 μm filter to exclude intact bacterialcells. Filtered supernatants may then be concentrated using methods thatmay include, but are not limited to, ammonium sulfate precipitation,ultracentrifugation, or filtration. For example, for ammonium sulfateprecipitation, 1.5-3 M ammonium sulfate can be added to filteredsupernatant slowly, while stirring at 4° C. Precipitations can beincubated at 4° C. for 8-48 hours and then centrifuged at 11,000×g for20-40 min at 4° C. The resulting pellets contain immune modulatingLactococcus strain EVs and other debris. Using ultracentrifugation,filtered supernatants can be centrifuged at 100,000-200,000×g for 1-16hours at 4° C. The pellet of this centrifugation contains immunemodulating Lactococcus strain EVs and other debris. In some embodiments,using a filtration technique, such as through the use of an Amicon Ultraspin filter or by tangential flow filtration, supernatants can befiltered so as to retain species of molecular weight >50 or 100 kDa.

Alternatively, EVs can be obtained from immune modulating Lactococcusstrain bacterial cultures continuously during growth, or at selectedtime points during growth, by connecting a bioreactor to an alternatingtangential flow (ATF) system (e.g., XCell ATF from Repligen). The ATFsystem retains intact cells (>0.22 um) in the bioreactor, and allowssmaller components (e.g., EVs, free proteins) to pass through a filterfor collection. For example, the system may be configured so that the<0.22 um filtrate is then passed through a second filter of 100 kDa,allowing species such as EVs between 0.22 um and 100 kDa to becollected, and species smaller than 100 kDa to be pumped back into thebioreactor. Alternatively, the system may be configured to allow formedium in the bioreactor to be replenished and/or modified during growthof the culture. EVs collected by this method may be further purifiedand/or concentrated by ultracentrifugation or filtration as describedabove for filtered supernatants.

EVs obtained by methods provided herein may be further purified by sizebased column chromatography, by affinity chromatography, and by gradientultracentrifugation, using methods that may include, but are not limitedto, use of a sucrose gradient or Optiprep gradient. Briefly, using asucrose gradient method, if ammonium sulfate precipitation orultracentrifugation were used to concentrate the filtered supernatants,pellets are resuspended in 60% sucrose, 30 mM Tris, pH 8.0. Iffiltration was used to concentrate the filtered supernatant, theconcentrate is buffer exchanged into 60% sucrose, 30 mM Tris, pH 8.0,using an Amicon Ultra column. Samples are applied to a 35-60%discontinuous sucrose gradient and centrifuged at 200,000×g for 3-24hours at 4° C. Briefly, using an Optiprep gradient method, if ammoniumsulfate precipitation or ultracentrifugation were used to concentratethe filtered supernatants, pellets are resuspended in 35% Optiprep inPBS. In some embodiments, if filtration was used to concentrate thefiltered supernatant, the concentrate is diluted using 60% Optiprep to afinal concentration of 35% Optiprep. Samples are applied to a 35-60%discontinuous sucrose gradient and centrifuged at 200,000×g for 3-24hours at 4° C.

In some embodiments, to confirm sterility and isolation of the EVpreparations, EVs are serially diluted onto agar medium used for routineculture of the bacteria being tested, and incubated using routineconditions. Non-sterile preparations are passed through a 0.22 um filterto exclude intact cells. To further increase purity, isolated EVs may beDNase or proteinase K treated.

In some embodiments, for preparation of EVs used for in vivo injections,purified EVs are processed as described previously (G. Norheim, et al.PLoS ONE. 10(9): e0134353 (2015)). Briefly, after sucrose gradientcentrifugation, bands containing EVs are resuspended to a finalconcentration of 50 μg/mL in a solution containing 3% sucrose or othersolution suitable for in vivo injection known to one skilled in the art.This solution may also contain adjuvant, for example aluminum hydroxideat a concentration of 0-0.5% (w/v).

In certain embodiments, to make samples compatible with further testing(e.g. to remove sucrose prior to TEM imaging or in vitro assays),samples are buffer exchanged into PBS or 30 mM Tris, pH 8.0 usingfiltration (e.g. Amicon Ultra columns), dialysis, or ultracentrifugation(200,000×g, ≥3 hours, 4° C.) and resuspension.

In some embodiments, the sterility of the EV preparations can beconfirmed by plating a portion of the EVs onto agar medium used forstandard culture of the bacteria used in the generation of the EVs andincubating using standard conditions.

In some embodiments select EVs are isolated and enriched bychromatography and binding surface moieties on EVs. In otherembodiments, select EVs are isolated and/or enriched by fluorescent cellsorting by methods using affinity reagents, chemical dyes, recombinantproteins or other methods known to one skilled in the art.

Bacterial Compositions

In certain aspects, provided herein are bacterial compositionscomprising an immune modulating Lactococcus strain provided herein, animmune modulating Lactococcus strain EVs provided herein, and/or animmune modulating Lactococcus strain PhAB provided herein. In someembodiments, the bacterial formulation further comprises apharmaceutically acceptable carrier.

In some embodiments, the bacterial composition comprises a killedbacterium, a live bacterium and/or an attenuated bacterium. Bacteria maybe heat-killed by pasteurization, sterilization, high temperaturetreatment, spray cooking and/or spray drying (heat treatments can beperformed at 50° C., 65° C., 85° C. or a variety of other temperaturesand/or a varied amount of time). Bacteria may also be killed orinactivated using γ-irradiation (gamma irradiation), exposure to UVlight, formalin-inactivation, and/or freezing methods, or a combinationthereof. For example, the bacteria may be exposed to 1, 2, 3, 4, 5, 10,15, 20, 25, 30, 35, 40, or 50 kGy of radiation prior to administration.In some embodiments, bacteria (e.g., Lactococcus strain) are killedusing gamma irradiation. In some embodiments, the bacteria are killed orinactivated using electron irradiation (e.g., beta radiation) or x-rayirradiation.

In certain embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the bacteria inthe bacterial composition are the immune modulating Lactococcus strain.In certain embodiments, substantially all of the bacteria in thebacterial composition are the immune modulating Lactococcus strain. Incertain embodiments, the bacterial composition comprises at least 1×10³colony forming units (CFUs), 1×10⁴ colony forming units (CFUs), 1×10⁵colony forming units (CFUs), 5×10⁵ colony forming units (CFUs), 1×10⁶colony forming units (CFUs), 2×10⁶ colony forming units (CFUs), 3×10⁶colony forming units (CFUs), 4×10⁶ colony forming units (CFUs), 5×10⁶colony forming units (CFUs), 6×10⁶ colony forming units (CFUs), 7×10⁶colony forming units (CFUs), 8×10⁶ colony forming units (CFUs), 9×10⁶colony forming units (CFUs), 1×10⁷ colony forming units (CFUs), 2×10⁷colony forming units (CFUs), 3×10⁷ colony forming units (CFUs), 4×10⁷colony forming units (CFUs), 5×10⁷ colony forming units (CFUs), 6×10⁷colony forming units (CFUs), 7×10⁷ colony forming units (CFUs), 8×10⁷colony forming units (CFUs), 9×10⁷ colony forming units (CFUs), 1×10⁸colony forming units (CFUs), 2×10⁸ colony forming units (CFUs), 3×10⁸colony forming units (CFUs), 4×10⁸ colony forming units (CFUs), 5×10⁸colony forming units (CFUs), 6×10⁸ colony forming units (CFUs), 7×10⁸colony forming units (CFUs), 8×10⁸ colony forming units (CFUs), 9×10⁸colony forming units (CFUs), 1×10⁹ colony forming units (CFUs), 5×10⁹colony forming units (CFUs), 1×10¹⁰ colony forming units (CFUs) 5×10¹⁰colony forming units (CFUs), 1×10¹¹ colony forming units (CFUs) 5×10¹¹colony forming units (CFUs), 1×10¹² colony forming units (CFUs) 5×10¹²colony forming units (CFUs), 1×10¹³ colony forming units (CFUs) of theimmune modulating Lactococcus strain.

In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%of the bacteria in the composition are of the immune modulatingLactococcus strain. 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of thebacteria in the composition are of the immune modulating Lactococcusstrain.

In some embodiments, the compositions described herein may include onlyone strains of the immune modulating Lactococcus described herein. Forexample, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19 or 20 of the immune modulating Lactococcus strains described herein,in any combination, can be included in the compositions provided herein.

In some embodiments, the pharmaceutical compositions comprise immunemodulating Lactococcus strain EVs substantially or entirely free ofbacteria. In some embodiments, the pharmaceutical compositions compriseboth immune modulating Lactococcus strain EVs and whole immunemodulating Lactococcus strain bacteria (e.g., live bacteria, killedbacteria, attenuated bacteria). In certain embodiments, thepharmaceutical compositions comprise immune modulating Lactococcusstrain bacteria that is substantially or entirely free of EVs.

In some embodiments, the pharmaceutical composition comprises at least 1immune modulating Lactococcus strain bacterium for every 1, 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2,4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2,7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, 10, 11, 12,13, 14, 15, 16, 17, 18. 19, 20, 21, 22, 23, 24, 25, 26, 27, 28. 29, 30,31, 32, 33, 34, 35, 36, 37, 38. 39, 40, 41, 42, 43, 44, 45, 46, 47, 48.49, 50, 51, 52, 53, 54, 55, 56, 57, 58. 59, 60, 61, 62, 63, 64, 65, 66,67, 68. 69, 70, 71, 72, 73, 74, 75, 76, 77, 78. 79, 80, 81, 82, 83, 84,85, 86, 87, 88. 89, 90, 91, 92, 93, 94, 95, 96, 97, 98. 99, 100, 150,200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850,900, 950, 1×10³, 2×10³, 3×10³, 4×10³, 5×10³, 6×10³, 7×10³, 8×10³, 9×10³,1×10⁴, 2×10⁴, 3×10⁴, 4×10⁴, 5×10⁴, 6×10⁴, 7×10⁴, 8×10⁴, 9×10⁴, 1×10⁵,2×10⁵, 3×10⁵, 4×10⁵, 5×10⁵, 6×10⁵, 7×10⁵, 8×10⁵, 9×10⁵, 1×10⁶, 2×10⁶,3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷,4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸,5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹,6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰,6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹,6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹, and/or 1×10¹² immune modulatingLactococcus strain EV particles.

In some embodiments, the pharmaceutical composition comprises about 1immune modulating Lactococcus strain bacterium for every 1, 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2,4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2,7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, 10, 11, 12,13, 14, 15, 16, 17, 18. 19, 20, 21, 22, 23, 24, 25, 26, 27, 28. 29, 30,31, 32, 33, 34, 35, 36, 37, 38. 39, 40, 41, 42, 43, 44, 45, 46, 47, 48.49, 50, 51, 52, 53, 54, 55, 56, 57, 58. 59, 60, 61, 62, 63, 64, 65, 66,67, 68. 69, 70, 71, 72, 73, 74, 75, 76, 77, 78. 79, 80, 81, 82, 83, 84,85, 86, 87, 88. 89, 90, 91, 92, 93, 94, 95, 96, 97, 98. 99, 100, 150,200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850,900, 950, 1×10³, 2×10³, 3×10³, 4×10³, 5×10³, 6×10³, 7×10³, 8×10³, 9×10³,1×10⁴, 2×10⁴, 3×10⁴, 4×10⁴, 5×10⁴, 6×10⁴, 7×10⁴, 8×10⁴, 9×10⁴, 1×10⁵,2×10⁵, 3×10⁵, 4×10⁵, 5×10⁵, 6×10⁵, 7×10⁵, 8×10⁵, 9×10⁵, 1×10⁶, 2×10⁶,3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷,4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸,5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹,6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰,6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹,6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹, and/or 1×10¹² immune modulatingLactococcus strain EV particles.

In certain embodiments, the pharmaceutical composition comprises acertain ratio of immune modulating Lactococcus strain bacteria particlesto immune modulating Lactococcus strain EV particles. The number ofimmune modulating Lactococcus strain bacteria particles can be based onactual particle number or (if the bacteria is live) the number of CFUs.The particle number can be established by combining a set number ofpurified immune modulating Lactococcus strain EVs with a set number ofpurified immune modulating Lactococcus strain bacterium, by modifyingthe growth conditions under which the immune modulating Lactococcusstrain bacteria are cultured, or by modifying the immune modulatingLactococcus strain bacteria itself to produce more or fewer immunemodulating Lactococcus strain EVs.

In some embodiments, to quantify the numbers of immune modulatingLactococcus strain EVs and/or immune modulating Lactococcus strainbacteria present in a bacterial sample, electron microscopy (e.g., EM ofultrathin frozen sections) can be used to visualize the vesicles andbacteria and count their relative numbers. Alternatively, combinationsof nanoparticle tracking analysis (NTA), Coulter counting, and dynamiclight scattering (DLS) or a combination of these techniques can be used.NTA and the Coulter counter count particles and show their sizes. DLSgives the size distribution of particles, but not the concentration.Bacteria frequently have diameters of 1-2 um. The full range is 0.2-20um. Combined results from Coulter counting and NTA can reveal thenumbers of bacteria in a given sample. Coulter counting reveals thenumbers of particles with diameters of 0.7-10 um. NTA reveals thenumbers of particles with diameters of 50-1400 nm. For most bacterialsamples, the Coulter counter alone can reveal the number of bacteria ina sample. EVs are 20-250 nm in diameter. NTA will allow us to count thenumbers of particles that are 50-250 nm in diameter. DLS reveals thedistribution of particles of different diameters within an approximaterange of 1 nm-3 um.

In some embodiments, the pharmaceutical composition comprises no morethan 1 immune modulating Lactococcus strain bacterium for every 1, 1.1,1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1,4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6,5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1,7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6,8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, 10, 11,12, 13, 14, 15, 16, 17, 18. 19, 20, 21, 22, 23, 24, 25, 26, 27, 28. 29,30, 31, 32, 33, 34, 35, 36, 37, 38. 39, 40, 41, 42, 43, 44, 45, 46, 47,48. 49, 50, 51, 52, 53, 54, 55, 56, 57, 58. 59, 60, 61, 62, 63, 64, 65,66, 67, 68. 69, 70, 71, 72, 73, 74, 75, 76, 77, 78. 79, 80, 81, 82, 83,84, 85, 86, 87, 88. 89, 90, 91, 92, 93, 94, 95, 96, 97, 98. 99, 100,150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800,850, 900, 950, 1×10³, 2×10³, 3×10³, 4×10³, 5×10³, 6×10³, 7×10³, 8×10³,9×10³, 1×10⁴, 2×10⁴, 3×10⁴, 4×10⁴, 5×10⁴, 6×10⁴, 7×10⁴, 8×10⁴, 9×10⁴,1×10⁵, 2×10⁵, 3×10⁵, 4×10⁵, 5×10⁵, 6×10⁵, 7×10⁵, 8×10⁵, 9×10⁵, 1×10⁶,2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷,3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸,4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹,5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰,5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹,5×10¹¹, 6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹, and/or 1×10¹² immune modulatingLactococcus strain EV particles.

In some embodiments, the pharmaceutical composition comprises at least 1immune modulating Lactococcus strain EV particle for every 1, 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2,4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2,7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, 10, 11, 12,13, 14, 15, 16, 17, 18. 19, 20, 21, 22, 23, 24, 25, 26, 27, 28. 29, 30,31, 32, 33, 34, 35, 36, 37, 38. 39, 40, 41, 42, 43, 44, 45, 46, 47, 48.49, 50, 51, 52, 53, 54, 55, 56, 57, 58. 59, 60, 61, 62, 63, 64, 65, 66,67, 68. 69, 70, 71, 72, 73, 74, 75, 76, 77, 78. 79, 80, 81, 82, 83, 84,85, 86, 87, 88. 89, 90, 91, 92, 93, 94, 95, 96, 97, 98. 99, 100, 150,200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850,900, 950, 1×10³, 2×10³, 3×10³, 4×10³, 5×10³, 6×10³, 7×10³, 8×10³, 9×10³,1×10⁴, 2×10⁴, 3×10⁴, 4×10⁴, 5×10⁴, 6×10⁴, 7×10⁴, 8×10⁴, 9×10⁴, 1×10⁵,2×10⁵, 3×10⁵, 4×10⁵, 5×10⁵, 6×10⁵, 7×10⁵, 8×10⁵, 9×10⁵, 1×10⁶, 2×10⁶,3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷,4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸,5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹,6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰,6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹,6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹, and/or 1×10¹² immune modulatingLactococcus strain bacterium.

In some embodiments, the pharmaceutical composition comprises about 1immune modulating Lactococcus strain EV particle for every 1, 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2,4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2,7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, 10, 11, 12,13, 14, 15, 16, 17, 18. 19, 20, 21, 22, 23, 24, 25, 26, 27, 28. 29, 30,31, 32, 33, 34, 35, 36, 37, 38. 39, 40, 41, 42, 43, 44, 45, 46, 47, 48.49, 50, 51, 52, 53, 54, 55, 56, 57, 58. 59, 60, 61, 62, 63, 64, 65, 66,67, 68. 69, 70, 71, 72, 73, 74, 75, 76, 77, 78. 79, 80, 81, 82, 83, 84,85, 86, 87, 88. 89, 90, 91, 92, 93, 94, 95, 96, 97, 98. 99, 100, 150,200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850,900, 950, 1×10³, 2×10³, 3×10³, 4×10³, 5×10³, 6×10³, 7×10³, 8×10³, 9×10³,1×10⁴, 2×10⁴, 3×10⁴, 4×10⁴, 5×10⁴, 6×10⁴, 7×10⁴, 8×10⁴, 9×10⁴, 1×10⁵,2×10⁵, 3×10⁵, 4×10⁵, 5×10⁵, 6×10⁵, 7×10⁵, 8×10⁵, 9×10⁵, 1×10⁶, 2×10⁶,3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷,4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸,5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹,6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰,6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹,6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹, and/or 1×10¹² immune modulatingLactococcus strain bacterium. In some embodiments, the pharmaceuticalcomposition comprises no more than 1 immune modulating Lactococcusstrain EV particle for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8.1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3,3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8.4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3,6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3,9.4, 9.5, 9.6, 9.7, 9.8. 9.9, 10, 11, 12, 13, 14, 15, 16, 17, 18. 19,20, 21, 22, 23, 24, 25, 26, 27, 28. 29, 30, 31, 32, 33, 34, 35, 36, 37,38. 39, 40, 41, 42, 43, 44, 45, 46, 47, 48. 49, 50, 51, 52, 53, 54, 55,56, 57, 58. 59, 60, 61, 62, 63, 64, 65, 66, 67, 68. 69, 70, 71, 72, 73,74, 75, 76, 77, 78. 79, 80, 81, 82, 83, 84, 85, 86, 87, 88. 89, 90, 91,92, 93, 94, 95, 96, 97, 98. 99, 100, 150, 200, 250, 300, 350, 400, 450,500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1×10³, 2×10³, 3×10³,4×10³, 5×10³, 6×10³, 7×10³, 8×10³, 9×10³, 1×10⁴, 2×10⁴, 3×10⁴, 4×10⁴,5×10⁴, 6×10⁴, 7×10⁴, 8×10⁴, 9×10⁴, 1×10⁵, 2×10⁵, 3×10⁵, 4×10⁵, 5×10⁵,6×10⁵, 7×10⁵, 8×10⁵, 9×10⁵, 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶,7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷,8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸,9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹,1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰,1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹, 6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹,and/or 1×10¹² immune modulating Lactococcus strain bacterium.

In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% of the particles in the pharmaceuticalcomposition are immune modulating Lactococcus strain EVs.

In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% of the particles in the pharmaceuticalcomposition are immune modulating Lactococcus strain bacteria.

In some embodiments, no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%,24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% of the particles in the pharmaceuticalcomposition are immune modulating Lactococcus strain EVs.

In some embodiments, no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%,24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% of the particles in the pharmaceuticalcomposition are immune modulating Lactococcus strain bacteria.

In some embodiments, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%,40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% of the particles in the pharmaceutical compositionare immune modulating Lactococcus strain EVs.

In some embodiments, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%,40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% of the particles in the pharmaceutical compositionare immune modulating Lactococcus strain bacteria.

In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% of the protein in the pharmaceuticalcomposition is immune modulating Lactococcus strain EV protein.

In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% of the protein in the pharmaceuticalcomposition is immune modulating Lactococcus strain bacteria protein.

In some embodiments, no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%,24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% of the protein in the pharmaceuticalcomposition is immune modulating Lactococcus strain EV protein.

In some embodiments, no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%,24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% of the protein in the pharmaceuticalcomposition is immune modulating Lactococcus strain bacteria protein.

In some embodiments, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%,40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% of the protein in the pharmaceutical composition isimmune modulating Lactococcus strain EV protein.

In some embodiments, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%,40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% of the protein in the pharmaceutical composition isimmune modulating Lactococcus strain bacteria protein.

In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% of the lipids in the pharmaceuticalcomposition are immune modulating Lactococcus strain EV lipids.

In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% of the lipids in the pharmaceuticalcomposition are immune modulating Lactococcus strain bacteria lipids.

In some embodiments, no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%,24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% of the lipids in the pharmaceuticalcomposition are immune modulating Lactococcus strain EV lipids.

In some embodiments, no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%,24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% of the lipids in the pharmaceuticalcomposition are immune modulating Lactococcus strain bacteria lipids.

In some embodiments, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%,40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% of the lipids in the pharmaceutical composition areimmune modulating Lactococcus strain EV lipids.

In some embodiments, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%,40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% of the lipids in the pharmaceutical composition areimmune modulating Lactococcus strain bacteria lipids.

In some embodiments, the immune modulating Lactococcus strain EVs in thepharmaceutical composition are purified from one or more other bacterialcomponents. In some embodiments, the pharmaceutical composition furthercomprises other bacterial components. In some embodiments, thepharmaceutical composition comprise bacteria cells.

As described in detail below, the pharmaceutical compositions disclosedherein may be specially formulated for administration in solid or liquidform, including those adapted for oral or rectal administration.

In some embodiments, the composition described herein may be apharmaceutical composition, a dietary supplement, or a food product(e.g., a food or beverage). In some embodiments, the food product is ananimal feed.

In certain embodiments, the pharmaceutical composition for oraladministration described herein comprises an additional component thatenables efficient delivery of the bacteria to the colon. In someembodiments, pharmaceutical preparation that enables the delivery of thebacteria to the colon can be used. Examples of such formulations includepH sensitive compositions, such as buffered sachet formulations orenteric polymers that release their contents when the pH becomesalkaline after the enteric polymers pass through the stomach. When a pHsensitive composition is used for formulating the pharmaceuticalpreparation, the pH sensitive composition can be a polymer whose pHthreshold of the decomposition of the composition is between about 6.8and about 7.5.

Another embodiment of a pharmaceutical composition useful for deliveryof the bacteria to the colon is one that ensures the delivery to thecolon by delaying the release of the bacteria by approximately 3 to 5hours, which corresponds to the small intestinal transit time. In someembodiments, the pharmaceutical composition for delayed release includesa hydrogel shell. The hydrogel is hydrated and swells upon contact withgastrointestinal fluid, with the result that the contents areeffectively released (released predominantly in the colon). Delayedrelease dosage units include bacteria-containing compositions having amaterial which coats or selectively coats the bacteria. Examples of sucha selective coating material include in vivo degradable polymers,gradually hydrolyzable polymers, gradually water-soluble polymers,and/or enzyme degradable polymers. A wide variety of coating materialsfor efficiently delaying the release is available and includes, forexample, cellulose-based polymers such as hydroxypropyl cellulose,acrylic acid polymers and copolymers such as methacrylic acid polymersand copolymers, and vinyl polymers and copolymers such aspolyvinylpyrrolidone.

Examples of composition enabling the delivery to the colon furtherinclude bioadhesive compositions which specifically adhere to thecolonic mucosal membrane (for example, a polymer described in thespecification of U.S. Pat. No. 6,368,586, hereby incorporated byreference) and compositions into which a protease inhibitor isincorporated for protecting particularly a biopharmaceutical preparationin the gastrointestinal tracts from decomposition due to an activity ofa protease.

An example of a system enabling the delivery to the colon is a system ofdelivering a composition to the colon by pressure change in such a waythat the contents are released by utilizing pressure change caused bygeneration of gas in bacterial fermentation at a distal portion of thestomach. Such a system is not particularly limited, and a more specificexample thereof is a capsule which has contents dispersed in asuppository base and which is coated with a hydrophobic polymer (forexample, ethyl cellulose).

Another example of the system enabling the delivery to the colon is asystem of delivering a composition to the colon, the system beingspecifically decomposed by an enzyme (for example, a carbohydratehydrolase or a carbohydrate reductase) present in the colon. Such asystem is not particularly limited, and more specific examples thereofinclude systems which use food components such as non-starchpolysaccharides, amylose, xanthan gum, and azopolymers.

In some embodiments, Probiotic formulations are provided asencapsulated, enteric coated, or powder forms, with doses ranging up to10¹¹ cfu (e.g., up to 10¹⁰ cfu). In some embodiments, the compositioncomprises 5×10¹¹ cfu of immune modulating Lactococcus strain and 10%(w/w) corn starch in a capsule. The capsule is enteric coated forduodenal release at pH 5.5 In some embodiments, the capsule is entericcoated for duodenal release at pH 5.5. In some embodiments, thecomposition comprises a powder of freeze-dried immune modulatingLactococcus strain which is deemed “Qualified Presumption of Safety”(QPS) status. In some embodiments, the composition is stable at frozenor refrigerated temperature.

Methods for producing microbial compositions may include three mainprocessing steps. The steps are: organism banking, organism production,and preservation. In certain embodiments, a sample that contains anabundance of immune modulating Lactococcus strain may be cultured byavoiding an isolation step.

For banking, the strains included in the microbial composition may be(1) isolated directly from a specimen or taken from a banked stock, (2)optionally cultured on a nutrient agar or broth that supports growth togenerate viable biomass, and (3) the biomass optionally preserved inmultiple aliquots in long-term storage.

In embodiments using a culturing step, the agar or broth may containnutrients that provide essential elements and specific factors thatenable growth. An example would be a medium composed of 20 g/L glucose,10 g/L yeast extract, 10 g/L soy peptone, 2 g/L citric acid, 1.5 g/Lsodium phosphate monobasic, 100 mg/L ferric ammonium citrate, 80 mg/Lmagnesium sulfate, 10 mg/L hemin chloride, 2 mg/L calcium chloride, 1mg/L menadione. Another example would be a medium composed of 10 g/Lbeef extract, 10 g/L peptone, 5 g/L sodium chloride, 5 g/L dextrose, 3g/L yeast extract, 3 g/L sodium acetate, 1 g/L soluble starch, and 0.5g/L L-cysteine HCl, at pH 6.8. A variety of microbiological media andvariations are well known in the art (e.g., R. M. Atlas, Handbook ofMicrobiological Media (2010) CRC Press). Culture media can be added tothe culture at the start, may be added during the culture, or may beintermittently/continuously flowed through the culture. The strains inthe bacterial composition may be cultivated alone, as a subset of themicrobial composition, or as an entire collection comprising themicrobial composition. As an example, a first strain may be cultivatedtogether with a second strain in a mixed continuous culture, at adilution rate lower than the maximum growth rate of either cell toprevent the culture from washing out of the cultivation.

The inoculated culture is incubated under favorable conditions for atime sufficient to build biomass. For microbial compositions for humanuse this is often at 37° C. temperature, pH, and other parameter withvalues similar to the normal human niche. The environment may beactively controlled, passively controlled (e.g., via buffers), orallowed to drift. For example, for anaerobic bacterial compositions, ananoxic/reducing environment may be employed. This can be accomplished byaddition of reducing agents such as cysteine to the broth, and/orstripping it of oxygen. As an example, a culture of a bacterialcomposition may be grown at 37° C., pH 7, in the medium above,pre-reduced with 1 g/L cysteine-HCl.

When the culture has generated sufficient biomass, it may be preservedfor banking. The organisms may be placed into a chemical milieu thatprotects from freezing (adding ‘cryoprotectants’), drying(‘lyoprotectants’), and/or osmotic shock (‘osmoprotectants’), dispensinginto multiple (optionally identical) containers to create a uniformbank, and then treating the culture for preservation. Containers aregenerally impermeable and have closures that assure isolation from theenvironment. Cryopreservation treatment is accomplished by freezing aliquid at ultra-low temperatures (e.g., at or below −80° C.). Driedpreservation removes water from the culture by evaporation (in the caseof spray drying or ‘cool drying’) or by sublimation (e.g., for freezedrying, spray freeze drying). Removal of water improves long-termmicrobial composition storage stability at temperatures elevated abovecryogenic conditions. If the microbial composition comprises, forexample, spore forming species and results in the production of spores,the final composition may be purified by additional means such asdensity gradient centrifugation and preserved using the techniques[?]described above[?]. Microbial composition banking may be done byculturing and preserving the strains individually, or by mixing thestrains together to create a combined bank. As an example ofcryopreservation, a microbial composition culture may be harvested bycentrifugation to pellet the cells from the culture medium, thesupernatant decanted and replaced with fresh culture broth containing15% glycerol. The culture can then be aliquoted into 1 mL cryotubes,sealed, and placed at −80° C. for long-term viability retention. Thisprocedure achieves acceptable viability upon recovery from frozenstorage.

Microbial production may be conducted using similar culture steps tobanking, including medium composition and culture conditions describedabove. It may be conducted at larger scales of operation, especially forclinical development or commercial production. At larger scales, theremay be several subcultivations of the microbial composition prior to thefinal cultivation. At the end of cultivation, the culture is harvestedto enable further formulation into a dosage form for administration.This can involve concentration, removal of undesirable mediumcomponents, and/or introduction into a chemical milieu that preservesthe microbial composition and renders it acceptable for administrationvia the chosen route. For example, a microbial composition may becultivated to a concentration of 10¹⁰ CFU/mL, then concentrated 20-foldby tangential flow microfiltration; the spent medium may be exchanged bydiafiltering with a preservative medium consisting of 2% gelatin, 100 mMtrehalose, and 10 mM sodium phosphate buffer. The suspension can then befreeze-dried to a powder and titrated.

After drying, the powder may be blended to an appropriate potency, andmixed with other cultures and/or a filler such as microcrystallinecellulose for consistency and ease of handling, and the bacterialcomposition formulated as provided herein.

In certain aspects, provided are bacterial compositions foradministration subjects. In some embodiments, the bacterial compositionsare combined with additional active and/or inactive materials in orderto produce a final product, which may be in single dosage unit or in amulti-dose format.

In some embodiments, the composition comprises at least onecarbohydrate. A “carbohydrate” refers to a sugar or polymer of sugars.The terms “saccharide,” “polysaccharide,” “carbohydrate,” and“oligosaccharide” may be used interchangeably. Most carbohydrates arealdehydes or ketones with many hydroxyl groups, usually one on eachcarbon atom of the molecule. Carbohydrates generally have the molecularformula C_(n)H_(2n)O_(n). A carbohydrate may be a monosaccharide, adisaccharide, trisaccharide, oligosaccharide, or polysaccharide. Themost basic carbohydrate is a monosaccharide, such as glucose, sucrose,galactose, mannose, ribose, arabinose, xylose, and fructose.Disaccharides are two joined monosaccharides. Exemplary disaccharidesinclude sucrose, maltose, cellobiose, and lactose. Typically, anoligosaccharide includes between three and six monosaccharide units(e.g., raffinose, stachyose), and polysaccharides include six or moremonosaccharide units. Exemplary polysaccharides include starch,glycogen, and cellulose. Carbohydrates may contain modified saccharideunits such as 2′-deoxyribose wherein a hydroxyl group is removed,2′-fluororibose wherein a hydroxyl group is replaced with a fluorine, orN-acetylglucosamine, a nitrogen-containing form of glucose (e.g.,2′-fluororibose, deoxyribose, and hexose). Carbohydrates may exist inmany different forms, for example, conformers, cyclic forms, acyclicforms, stereoisomers, tautomers, anomers, and isomers.

In some embodiments, the composition comprises at least one lipid. Asused herein, a “lipid” includes fats, oils, triglycerides, cholesterol,phospholipids, fatty acids in any form including free fatty acids. Fats,oils and fatty acids can be saturated, unsaturated (cis or trans) orpartially unsaturated (cis or trans). In some embodiments the lipidcomprises at least one fatty acid selected from lauric acid (12:0),myristic acid (14:0), palmitic acid (16:0), palmitoleic acid (16:1),margaric acid (17:0), heptadecenoic acid (17:1), stearic acid (18:0),oleic acid (18:1), linoleic acid (18:2), linolenic acid (18:3),octadecatetraenoic acid (18:4), arachidic acid (20:0), eicosenoic acid(20:1), eicosadienoic acid (20:2), eicosatetraenoic acid (20:4),eicosapentaenoic acid (20:5) (EPA), docosanoic acid (22:0), docosenoicacid (22:1), docosapentaenoic acid (22:5), docosahexaenoic acid (22:6)(DHA), and tetracosanoic acid (24:0). In some embodiments thecomposition comprises at least one modified lipid, for example a lipidthat has been modified by cooking.

In some embodiments, the composition comprises at least one supplementalmineral or mineral source. Examples of minerals include, withoutlimitation: chloride, sodium, calcium, iron, chromium, copper, iodine,zinc, magnesium, manganese, molybdenum, phosphorus, potassium, andselenium. Suitable forms of any of the foregoing minerals includesoluble mineral salts, slightly soluble mineral salts, insoluble mineralsalts, chelated minerals, mineral complexes, non-reactive minerals suchas carbonyl minerals, and reduced minerals, and combinations thereof.

In some embodiments, the composition comprises at least one supplementalvitamin. The at least one vitamin can be fat-soluble or water solublevitamins. Suitable vitamins include but are not limited to vitamin C,vitamin A, vitamin E, vitamin B12, vitamin K, riboflavin, niacin,vitamin D, vitamin B6, folic acid, pyridoxine, thiamine, pantothenicacid, and biotin. Suitable forms of any of the foregoing are salts ofthe vitamin, derivatives of the vitamin, compounds having the same orsimilar activity of the vitamin, and metabolites of the vitamin.

In some embodiments, the composition comprises an excipient.Non-limiting examples of suitable excipients include a buffering agent,a preservative, a stabilizer, a binder, a compaction agent, a lubricant,a dispersion enhancer, a disintegration agent, a flavoring agent, asweetener, and a coloring agent.

In some embodiments, the excipient is a buffering agent. Non-limitingexamples of suitable buffering agents include sodium citrate, magnesiumcarbonate, magnesium bicarbonate, calcium carbonate, and calciumbicarbonate.

In some embodiments, the excipient comprises a preservative.Non-limiting examples of suitable preservatives include antioxidants,such as alpha-tocopherol and ascorbate, and antimicrobials, such asparabens, chlorobutanol, and phenol.

In some embodiments, the composition comprises a binder as an excipient.Non-limiting examples of suitable binders include starches,pregelatinized starches, gelatin, polyvinylpyrolidone, cellulose,methylcellulose, sodium carboxymethylcellulose, ethylcellulose,polyacrylamides, polyvinyloxoazolidone, polyvinylalcohols, C₁₂-C₁₈ fattyacid alcohol, polyethylene glycol, polyols, saccharides,oligosaccharides, and combinations thereof.

In some embodiments, the composition comprises a lubricant as anexcipient. Non-limiting examples of suitable lubricants includemagnesium stearate, calcium stearate, zinc stearate, hydrogenatedvegetable oils, sterotex, polyoxyethylene monostearate, talc,polyethyleneglycol, sodium benzoate, sodium lauryl sulfate, magnesiumlauryl sulfate, and light mineral oil.

In some embodiments, the composition comprises a dispersion enhancer asan excipient. Non-limiting examples of suitable dispersants includestarch, alginic acid, polyvinylpyrrolidones, guar gum, kaolin,bentonite, purified wood cellulose, sodium starch glycolate,isoamorphous silicate, and microcrystalline cellulose as high HLBemulsifier surfactants.

In some embodiments, the composition comprises a disintegrant as anexcipient. In some embodiments the disintegrant is a non-effervescentdisintegrant. Non-limiting examples of suitable non-effervescentdisintegrants include starches such as corn starch, potato starch,pregelatinized and modified starches thereof, sweeteners, clays, such asbentonite, microcrystalline cellulose, alginates, sodium starchglycolate, gums such as agar, guar, locust bean, karaya, pectin, andtragacanth. In some embodiments the disintegrant is an effervescentdisintegrant. Non-limiting examples of suitable effervescentdisintegrants include sodium bicarbonate in combination with citricacid, and sodium bicarbonate in combination with tartaric acid.

In some embodiments, the bacterial formulation comprises an entericcoating or micro encapsulation. In certain embodiments, the entericcoating or micro encapsulation improves targeting to a desired region ofthe gastrointestinal tract. For example, in certain embodiments, thebacterial composition comprises an enteric coating and/or microcapsulesthat dissolves at a pH associated with a particular region of thegastrointestinal tract. In some embodiments, the enteric coating and/ormicrocapsules dissolve at a pH of about 5.5-6.2 to release in theduodenum, at a pH value of about 7.2-7.5 to release in the ileum, and/orat a pH value of about 5.6-6.2 to release in the colon. Exemplaryenteric coatings and microcapsules are described, for example, in U.S.Pat. Pub. No. 2016/0022592, which is hereby incorporated by reference inits entirety.

In some embodiments, the composition is a food product (e.g., a food orbeverage) such as a health food or beverage, a food or beverage forinfants, a food or beverage for pregnant women, athletes, seniorcitizens or other specified group, a functional food, a beverage, a foodor beverage for specified health use, a dietary supplement, a food orbeverage for patients, or an animal feed. Specific examples of the foodsand beverages include various beverages such as juices, refreshingbeverages, tea beverages, drink preparations, jelly beverages, andfunctional beverages; alcoholic beverages such as beers;carbohydrate-containing foods such as rice food products, noodles,breads, and pastas; paste products such as fish hams, sausages, pasteproducts of seafood; retort pouch products such as curries, food dressedwith a thick starchy sauces, and Chinese soups; soups; dairy productssuch as milk, dairy beverages, ice creams, cheeses, and yogurts;fermented products such as fermented soybean pastes, yogurts, fermentedbeverages, and pickles; bean products; various confectionery products,including biscuits, cookies, and the like, candies, chewing gums,gummies, cold desserts including jellies, cream caramels, and frozendesserts; instant foods such as instant soups and instant soy-beansoups; microwavable foods; and the like. Further, the examples alsoinclude health foods and beverages prepared in the forms of powders,granules, tablets, capsules, liquids, pastes, and jellies.

In certain embodiments, the bacteria disclosed herein are administeredin conjunction with a prebiotic to the subject. Prebiotics arecarbohydrates which are generally indigestible by a host animal and areselectively fermented or metabolized by bacteria. Prebiotics may beshort-chain carbohydrates (e.g., oligosaccharides) and/or simple sugars(e.g., mono- and di-saccharides) and/or mucins (heavily glycosylatedproteins) that alter the composition or metabolism of a microbiome inthe host. The short chain carbohydrates are also referred to asoligosaccharides, and usually contain from 2 or 3 and up to 8, 9, 10, 15or more sugar moieties. When prebiotics are introduced to a host, theprebiotics affect the bacteria within the host and do not directlyaffect the host. In certain aspects, a prebiotic composition canselectively stimulate the growth and/or activity of one of a limitednumber of bacteria in a host. Prebiotics include oligosaccharides suchas fructooligosaccharides (FOS) (including inulin),galactooligosaccharides (GOS), trans-galactooligosaccharides,xylooligosaccharides (XOS), chitooligosaccharides (COS), soyoligosaccharides (e.g., stachyose and raffinose) gentiooligosaccharides,isomaltooligosaccharides, mannooligosaccharides, maltooligosaccharidesand mannanoligosaccharides. Oligosaccharides are not necessarily singlecomponents, and can be mixtures containing oligosaccharides withdifferent degrees of oligomerization, sometimes including the parentdisaccharide and the monomeric sugars. Various types of oligosaccharidesare found as natural components in many common foods, including fruits,vegetables, milk, and honey. Specific examples of oligosaccharides arelactulose, lactosucrose, palatinose, glycosyl sucrose, guar gum, gumArabic, tagalose, amylose, amylopectin, pectin, xylan, andcyclodextrins. Prebiotics may also be purified or chemically orenzymatically synthesized.

Production of PhABs

In certain aspects, the PhABs described herein can be prepared using anymethod known in the art.

In some embodiments, the PhABs described herein are prepared byfractionation. Bacterial cells and/or supernatants from culturedbacteria cells are fractionated into various pharmacologically activebiomass (PhABs) and/or products derived therefrom. Bacterial cellsand/or supernatants are fractionated using materials and methods knownin the art (see e.g. Sandrini et al. Fractionation byultracentrifugation of gram negative cytoplasmic and membrane proteins.2014. Bio-Protocol. 4(21); Scholler et al. Protoplast and cytoplasmicmembrane preparations from Streptococcus sanguis and Streptococcusmutans. 1983. J Gen Micro. 129: 3271-3279; Thein et al. Efficientsubfractionation of gram-negative bacteria for proteomics studies. 2010.Am Chem Society. 9: 6135-6147; Hobb et al. Evaluation of procedures forouter membrane isolation from Campylobacter jejuni. 2009. 155(Pt. 3):979-988).

Additionally, PhABs obtained by methods provided herein may be furtherpurified by size based column chromatography, by affinitychromatography, and by gradient ultracentrifugation, using methods thatmay include, but are not limited to, use of a sucrose gradient orOptiprep gradient. Briefly, using a sucrose gradient method, if ammoniumsulfate precipitation or ultracentrifugation were used to concentratethe filtered supernatants, pellets are resuspended in 60% sucrose, 30 mMTris, pH 8.0. If filtration was used to concentrate the filteredsupernatant, the concentrate is buffer exchanged into 60% sucrose, 30 mMTris, pH 8.0, using an Amicon Ultra column. Samples are applied to a35-60% discontinuous sucrose gradient and centrifuged at 200,000×g for3-24 hours at 4° C. Briefly, using an Optiprep gradient method, ifammonium sulfate precipitation or ultracentrifugation were used toconcentrate the filtered supernatants, pellets are resuspended in 35%Optiprep in PBS. In some embodiments, if filtration was used toconcentrate the filtered supernatant, the concentrate is diluted using60% Optiprep to a final concentration of 35% Optiprep. Samples areapplied to a 35-60% discontinuous sucrose gradient and centrifuged at200,000×g for 3-24 hours at 4° C.

In some embodiments, to confirm sterility and isolation of the PhABpreparations, PhABs are serially diluted onto agar medium used forroutine culture of the bacteria being tested, and incubated usingroutine conditions. Non-sterile preparations are passed through a 0.22um filter to exclude intact cells. To further increase purity, isolatedPhABs may be DNase or proteinase K treated.

In some embodiments, for preparation of PhABs used for in vivoinjections, purified PhABs are processed as described previously (G.Norheim, et al. PLoS ONE. 10(9): e0134353 (2015)). Briefly, aftersucrose gradient centrifugation, bands containing PhABs are resuspendedto a final concentration of 50 μg/mL in a solution containing 3% sucroseor other solution suitable for in vivo injection known to one skilled inthe art. This solution may also contain adjuvant, for example aluminumhydroxide at a concentration of 0-0.5% (w/v).

In certain embodiments, to make samples compatible with further testing(e.g. to remove sucrose prior to TEM imaging or in vitro assays),samples are buffer exchanged into PBS or 30 mM Tris, pH 8.0 usingfiltration (e.g. Amicon Ultra columns), dialysis, or ultracentrifugation(200,000×g, ≥3 hours, 4° C.) and resuspension.

In some embodiments, the sterility of the PhAB preparations can beconfirmed by plating a portion of the PhABs onto agar medium used forstandard culture of the bacteria used in the generation of the PhABs andincubating using standard conditions.

In some embodiments select PhABs are isolated and enriched bychromatography and binding surface moieties on PhABs. In otherembodiments, select PhABs are isolated and/or enriched by fluorescentcell sorting by methods using affinity reagents, chemical dyes,recombinant proteins or other methods known to one skilled in the art.

Administration

In certain aspects, provided herein is a method of delivering abacterium and/or a bacterial composition described herein to a subject.In some embodiments of the methods provided herein, the bacteria areadministered in conjunction with the administration of an additionaltherapeutic. In some embodiments, the bacteria is co-formulated in apharmaceutical composition with the additional therapeutic. In someembodiments, the bacteria is co-administered with the additionaltherapeutic. In some embodiments, the additional therapeutic isadministered to the subject before administration of the bacteria (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or55 minutes before, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22 or 23 hours before, or about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days before). In some embodiments,the additional therapeutic is administered to the subject afteradministration of the bacteria (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25, 30, 35, 40, 45, 50 or 55 minutes after, about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or23 hours after, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14days after). In some embodiments, the same mode of delivery is used todeliver both the bacteria and the additional therapeutic. In someembodiments different modes of delivery are used to administer thebacteria and the additional therapeutic. For example, in someembodiments, the bacteria is administered orally while the additionaltherapeutic is administered via injection (e.g., an intravenous,intramuscular and/or intratumoral injection).

In certain embodiments, the pharmaceutical compositions, dosage forms,and kits described herein can be administered in conjunction with anyother conventional anti-immune disorder treatment. These treatments maybe applied as necessary and/or as indicated and may occur before,concurrent with or after administration of the pharmaceuticalcompositions, dosage forms, and kits described herein.

The dosage regimen can be any of a variety of methods and amounts, andcan be determined by one skilled in the art according to known clinicalfactors. As is known in the medical arts, dosages for any one patientcan depend on many factors, including the subject's species, size, bodysurface area, age, sex, immunocompetence, and general health, theparticular microorganism to be administered, duration and route ofadministration, the kind and stage of the disease, for example, tumorsize, and other compounds such as drugs being administered concurrently.In addition to the above factors, such levels can be affected by theinfectivity of the microorganism, and the nature of the microorganism,as can be determined by one skilled in the art. In the present methods,appropriate minimum dosage levels of microorganisms can be levelssufficient for the microorganism to survive, grow and replicate. Themethods of treatment described herein may be suitable for the treatmentof an immune disorder (e.g., an autoimmune disease, an inflammatorydisease, an allergy). The dose of the pharmaceutical compositionsdescribed herein may be appropriately set or adjusted in accordance withthe dosage form, the route of administration, the degree or stage of atarget disease, and the like. For example, the general effective dose ofthe agents may range between 0.01 mg/kg body weight/day and 1000 mg/kgbody weight/day, between 0.1 mg/kg body weight/day and 1000 mg/kg bodyweight/day, 0.5 mg/kg body weight/day and 500 mg/kg body weight/day, 1mg/kg body weight/day and 100 mg/kg body weight/day, or between 5 mg/kgbody weight/day and 50 mg/kg body weight/day. The effective dose may be0.01, 0.05, 0.1, 0.5, 1, 2, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 200, 500, or 1000 mg/kg body weight/day or more, but the dose isnot limited thereto.

In some embodiments, the dose administered to a subject is sufficient toprevent the immune disorder, delay its onset, or slow or stop itsprogression or prevent a relapse of the immune disorder. One skilled inthe art will recognize that dosage will depend upon a variety of factorsincluding the strength of the particular compound employed, as well asthe age, species, condition, and body weight of the subject. The size ofthe dose will also be determined by the route, timing, and frequency ofadministration as well as the existence, nature, and extent of anyadverse side-effects that might accompany the administration of aparticular compound and the desired physiological effect.

Suitable doses and dosage regimens can be determined by conventionalrange-finding techniques known to those of ordinary skill in the art.Generally, treatment is initiated with smaller dosages, which are lessthan the optimum dose of the compound. Thereafter, the dosage isincreased by small increments until the optimum effect under thecircumstances is reached. An effective dosage and treatment protocol canbe determined by routine and conventional means, starting e.g., with alow dose in laboratory animals and then increasing the dosage whilemonitoring the effects, and systematically varying the dosage regimen aswell. Animal studies are commonly used to determine the maximaltolerable dose (“MTD”) of bioactive agent per kilogram weight. Thoseskilled in the art regularly extrapolate doses for efficacy, whileavoiding toxicity, in other species, including humans.

In accordance with the above, in therapeutic applications, the dosagesof the active agents used in accordance with the invention varydepending on the active agent, the age, weight, and clinical conditionof the recipient patient, and the experience and judgment of theclinician or practitioner administering the therapy, among other factorsaffecting the selected dosage. Generally, the dose should be sufficientto result in slowing, and preferably regressing, the advancement of animmune disorder.

Separate administrations can include any number of two or moreadministrations (e.g., doses), including two, three, four, five or sixadministrations. One skilled in the art can readily determine the numberof administrations to perform, or the desirability of performing one ormore additional administrations, according to methods known in the artfor monitoring therapeutic methods and other monitoring methods providedherein. In some embodiments, the doses may be separated by at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29 or 30 days or 1, 2, 3, or 4 weeks.Accordingly, the methods provided herein include methods of providing tothe subject one or more administrations of a bacterium, where the numberof administrations can be determined by monitoring the subject, and,based on the results of the monitoring, determining whether or not toprovide one or more additional administrations. Deciding on whether ornot to provide one or more additional administrations can be based on avariety of monitoring results, including, but not limited to, indicationof tumor growth or inhibition of tumor growth, appearance of newmetastases or inhibition of metastasis, the subject's anti-bacteriumantibody titer, the subject's anti-tumor antibody titer, the overallhealth of the subject and/or the weight of the subject.

The time period between administrations can be any of a variety of timeperiods. The time period between administrations can be a function ofany of a variety of factors, including monitoring steps, as described inrelation to the number of administrations, the time period for a subjectto mount an immune response and/or the time period for a subject toclear the bacteria from normal tissue. In one example, the time periodcan be a function of the time period for a subject to mount an immuneresponse; for example, the time period can be more than the time periodfor a subject to mount an immune response, such as more than about oneweek, more than about ten days, more than about two weeks, or more thanabout a month; in another example, the time period can be less than thetime period for a subject to mount an immune response, such as less thanabout one week, less than about ten days, less than about two weeks, orless than about a month. In another example, the time period can be afunction of the time period for a subject to clear the bacteria fromnormal tissue; for example, the time period can be more than the timeperiod for a subject to clear the bacteria from normal tissue, such asmore than about a day, more than about two days, more than about threedays, more than about five days, or more than about a week.

In some embodiments, the delivery of an immune disorder therapeutic incombination with the bacteria described herein reduces the adverseeffects and/or improves the efficacy of the immune disorder therapeutic.

The effective dose of an immune disorder therapeutic described herein isthe amount of the therapeutic agent that is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, with the least toxicity to the patient. Theeffective dosage level can be identified using the methods describedherein and will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions administered, theroute of administration, the time of administration, the rate ofexcretion of the particular compound being employed, the duration of thetreatment, other drugs, compounds and/or materials used in combinationwith the particular compositions employed, the age, sex, weight,condition, general health and prior medical history of the patient beingtreated, and like factors well known in the medical arts. In general, aneffective dose of an immune disorder therapy will be the amount of thetherapeutic agent, which is the lowest dose effective to produce atherapeutic effect. Such an effective dose will generally depend uponthe factors described above.

The toxicity of an immune disorder therapy is the level of adverseeffects experienced by the subject during and following treatment.Adverse events associated with immune disorder therapy toxicity include,but are not limited to, abdominal pain, acid indigestion, acid reflux,allergic reactions, alopecia, anaphylaxis, anemia, anxiety, lack ofappetite, arthralgias, asthenia, ataxia, azotemia, loss of balance, bonepain, bleeding, blood clots, low blood pressure, elevated bloodpressure, difficulty breathing, bronchitis, bruising, low white bloodcell count, low red blood cell count, low platelet count,cardiotoxicity, cystitis, hemorrhagic cystitis, arrhythmias, heart valvedisease, cardiomyopathy, coronary artery disease, cataracts, centralneurotoxicity, cognitive impairment, confusion, conjunctivitis,constipation, coughing, cramping, cystitis, deep vein thrombosis,dehydration, depression, diarrhea, dizziness, dry mouth, dry skin,dyspepsia, dyspnea, edema, electrolyte imbalance, esophagitis, fatigue,loss of fertility, fever, flatulence, flushing, gastric reflux,gastroesophageal reflux disease, genital pain, granulocytopenia,gynecomastia, glaucoma, hair loss, hand-foot syndrome, headache, hearingloss, heart failure, heart palpitations, heartburn, hematoma,hemorrhagic cystitis, hepatotoxicity, hyperamylasemia, hypercalcemia,hyperchloremia, hyperglycemia, hyperkalemia, hyperlipasemia,hypermagnesemia, hypernatremia, hyperphosphatemia, hyperpigmentation,hypertriglyceridemia, hyperuricemia, hypoalbuminemia, hypocalcemia,hypochloremia, hypoglycemia, hypokalemia, hypomagnesemia, hyponatremia,hypophosphatemia, impotence, infection, injection site reactions,insomnia, iron deficiency, itching, joint pain, kidney failure,leukopenia, liver dysfunction, memory loss, menopause, mouth sores,mucositis, muscle pain, myalgias, myelosuppression, myocarditis,neutropenic fever, nausea, nephrotoxicity, neutropenia, nosebleeds,numbness, ototoxicity, pain, palmar-plantar erythrodysesthesia,pancytopenia, pericarditis, peripheral neuropathy, pharyngitis,photophobia, photosensitivity, pneumonia, pneumonitis, proteinuria,pulmonary embolus, pulmonary fibrosis, pulmonary toxicity, rash, rapidheart beat, rectal bleeding, restlessness, rhinitis, seizures, shortnessof breath, sinusitis, thrombocytopenia, tinnitus, urinary tractinfection, vaginal bleeding, vaginal dryness, vertigo, water retention,weakness, weight loss, weight gain, and xerostomia. In general, toxicityis acceptable if the benefits to the subject achieved through thetherapy outweigh the adverse events experienced by the subject due tothe therapy.

In some embodiments, the administration of the bacterial compositiontreats the immune disorder.

Therapeutic Agents

In certain aspects, the methods provided herein include theadministration to a subject of a bacterium and/or a bacterialcomposition described herein (e.g., an immune modulating Lactococcusstrain-containing bacterial composition) either alone or in combinationwith another therapeutic. In some embodiments, the bacterial compositionand the other therapy can be administered to the subject in any order.In some embodiments, the bacterial composition and the other therapy areadministered conjointly.

In some embodiments the bacterium is administered to the subject beforethe additional therapeutic is administered (e.g., at least 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or24 hours before or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30days before). In some embodiments the bacterium is administered to thesubject after the additional therapeutic is administered (e.g., at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23 or 24 hours after or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29 or 30 days after). In some embodiments, the bacterium and theadditional therapeutic are administered to the subject simultaneously ornearly simultaneously (e.g., administrations occur within an hour ofeach other). In some embodiments, the subject is administered anantibiotic before the bacterium is administered to the subject (e.g., atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23 or 24 hours before or at least 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29 or 30 days before). In some embodiments, the subject isadministered an antibiotic after the bacterium is administered to thesubject (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours before or at least 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29 or 30 days after). In some embodiments, thebacterium and the antibiotic are administered to the subjectsimultaneously or nearly simultaneously (e.g., administrations occurwithin an hour of each other).

In certain embodiments, the subject may undergo surgery. Types ofsurgery include but are not limited to preventative, diagnostic orstaging, curative and palliative surgery.

In some embodiments, the additional therapeutic is an antibiotic. Forexample, if the presence of a immune-disorder-associated bacteria and/oran immune-disorder-associated microbiome profile is detected accordingto the methods provided herein, antibiotics can be administered toeliminate the immune-disorder-associated bacteria from the subject.“Antibiotics” broadly refers to compounds capable of inhibiting orpreventing a bacterial infection. Antibiotics can be classified in anumber of ways, including their use for specific infections, theirmechanism of action, their bioavailability, or their spectrum of targetmicrobe (e.g., Gram-negative vs. Gram-positive bacteria, aerobic vs.anaerobic bacteria, etc.) and these may be used to kill specificbacteria in specific areas of the host (“niches”) (Leekha, et al 2011.General Principles of Antimicrobial Therapy. Mayo Clin Proc. 86(2):156-167). In certain embodiments, antibiotics can be used to selectivelytarget bacteria of a specific niche. In some embodiments, antibioticsknown to treat a particular infection that includes an immune disorderniche may be used to target immune-disorder-associated microbes,including immune-disorder-associated bacteria in that niche. In otherembodiments, antibiotics are administered after the bacterial treatment.In some embodiments, antibiotics are administered after the bacterialtreatment to remove the engraftment.

In some aspects, antibiotics can be selected based on their bactericidalor bacteriostatic properties. Bactericidal antibiotics includemechanisms of action that disrupt the cell wall (e.g., β-lactams), thecell membrane (e.g., daptomycin), or bacterial DNA (e.g.,fluoroquinolones). Bacteriostatic agents inhibit bacterial replicationand include sulfonamides, tetracyclines, and macrolides, and act byinhibiting protein synthesis. Furthermore, while some drugs can bebactericidal in certain organisms and bacteriostatic in others, knowingthe target organism allows one skilled in the art to select anantibiotic with the appropriate properties. In certain treatmentconditions, bacteriostatic antibiotics inhibit the activity ofbactericidal antibiotics. Thus, in certain embodiments, bactericidal andbacteriostatic antibiotics are not combined.

Antibiotics include, but are not limited to aminoglycosides, ansamycins,carbacephems, carbapenems, cephalosporins, glycopeptides, lincosamides,lipopeptides, macrolides, monobactams, nitrofurans, oxazolidonones,penicillins, polypeptide antibiotics, quinolones, fluoroquinolone,sulfonamides, tetracyclines, and anti-mycobacterial compounds, andcombinations thereof.

Aminoglycosides include, but are not limited to Amikacin, Gentamicin,Kanamycin, Neomycin, Netilmicin, Tobramycin, Paromomycin, andSpectinomycin. Aminoglycosides are effective, e.g., againstGram-negative bacteria, such as Escherichia coli, Klebsiella,Pseudomonas aeruginosa, and Francisella tularensis, and against certainaerobic bacteria but less effective against obligate/facultativeanaerobes. Aminoglycosides are believed to bind to the bacterial 30S or50S ribosomal subunit thereby inhibiting bacterial protein synthesis.

Ansamycins include, but are not limited to, Geldanamycin, Herbimycin,Rifamycin, and Streptovaricin. Geldanamycin and Herbimycin are believedto inhibit or alter the function of Heat Shock Protein 90.

Carbacephems include, but are not limited to, Loracarbef. Carbacephemsare believed to inhibit bacterial cell wall synthesis.

Carbapenems include, but are not limited to, Ertapenem, Doripenem,Imipenem/Cilastatin, and Meropenem. Carbapenems are bactericidal forboth Gram-positive and Gram-negative bacteria as broad-spectrumantibiotics. Carbapenems are believed to inhibit bacterial cell wallsynthesis.

Cephalosporins include, but are not limited to, Cefadroxil, Cefazolin,Cefalotin, Cefalothin, Cefalexin, Cefaclor, Cefamandole, Cefoxitin,Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefditoren, Cefoperazone,Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten, Ceftizoxime,Ceftriaxone, Cefepime, Ceftaroline fosamil, and Ceftobiprole. SelectedCephalosporins are effective, e.g., against Gram-negative bacteria andagainst Gram-positive bacteria, including Pseudomonas, certainCephalosporins are effective against methicillin-resistantStaphylococcus aureus (MRSA). Cephalosporins are believed to inhibitbacterial cell wall synthesis by disrupting synthesis of thepeptidoglycan layer of bacterial cell walls.

Glycopeptides include, but are not limited to, Teicoplanin, Vancomycin,and Telavancin. Glycopeptides are effective, e.g., against aerobic andanaerobic Gram-positive bacteria including MRSA and Clostridiumdifficile. Glycopeptides are believed to inhibit bacterial cell wallsynthesis by disrupting synthesis of the peptidoglycan layer ofbacterial cell walls.

Lincosamides include, but are not limited to, Clindamycin andLincomycin. Lincosamides are effective, e.g., against anaerobicbacteria, as well as Staphylococcus, and Streptococcus. Lincosamides arebelieved to bind to the bacterial 50S ribosomal subunit therebyinhibiting bacterial protein synthesis.

Lipopeptides include, but are not limited to, Daptomycin. Lipopeptidesare effective, e.g., against Gram-positive bacteria. Lipopeptides arebelieved to bind to the bacterial membrane and cause rapiddepolarization.

Macrolides include, but are not limited to, Azithromycin,Clarithromycin, Dirithromycin, Erythromycin, Roxithromycin,Troleandomycin, Telithromycin, and Spiramycin. Macrolides are effective,e.g., against Streptococcus and Mycoplasma. Macrolides are believed tobind to the bacterial or 50S ribosomal subunit, thereby inhibitingbacterial protein synthesis.

Monobactams include, but are not limited to, Aztreonam. Monobactams areeffective, e.g., against Gram-negative bacteria. Monobactams arebelieved to inhibit bacterial cell wall synthesis by disruptingsynthesis of the peptidoglycan layer of bacterial cell walls.

Nitrofurans include, but are not limited to, Furazolidone andNitrofurantoin.

Oxazolidonones include, but are not limited to, Linezolid, Posizolid,Radezolid, and Torezolid. Oxazolidonones are believed to be proteinsynthesis inhibitors.

Penicillins include, but are not limited to, Amoxicillin, Ampicillin,Azlocillin, Carbenicillin, Cloxacillin, Dicloxacillin, Flucloxacillin,Mezlocillin, Methicillin, Nafcillin, Oxacillin, Penicillin G, PenicillinV, Piperacillin, Temocillin and Ticarcillin. Penicillins are effective,e.g., against Gram-positive bacteria, facultative anaerobes, e.g.,Streptococcus, Borrelia, and Treponema. Penicillins are believed toinhibit bacterial cell wall synthesis by disrupting synthesis of thepeptidoglycan layer of bacterial cell walls.

Penicillin combinations include, but are not limited to,Amoxicillin/clavulanate, Ampicillin/sulbactam, Piperacillin/tazobactam,and Ticarcillin/clavulanate.

Polypeptide antibiotics include, but are not limited to, Bacitracin,Colistin, and Polymyxin B and E. Polypeptide Antibiotics are effective,e.g., against Gram-negative bacteria. Certain polypeptide antibioticsare believed to inhibit isoprenyl pyrophosphate involved in synthesis ofthe peptidoglycan layer of bacterial cell walls, while othersdestabilize the bacterial outer membrane by displacing bacterialcounter-ions.

Quinolones and Fluoroquinolone include, but are not limited to,Ciprofloxacin, Enoxacin, Gatifloxacin, Gemifloxacin, Levofloxacin,Lomefloxacin, Moxifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin,Trovafloxacin, Grepafloxacin, Sparfloxacin, and Temafloxacin.Quinolones/Fluoroquinolone are effective, e.g., against Streptococcusand Neisseria. Quinolones/Fluoroquinolone are believed to inhibit thebacterial DNA gyrase or topoisomerase IV, thereby inhibiting DNAreplication and transcription.

Sulfonamides include, but are not limited to, Mafenide, Sulfacetamide,Sulfadiazine, Silver sulfadiazine, Sulfadimethoxine, Sulfamethizole,Sulfamethoxazole, Sulfanilimide, Sulfasalazine, Sulfisoxazole,Trimethoprim-Sulfamethoxazole (Co-trimoxazole), andSulfonamidochrysoidine. Sulfonamides are believed to inhibit folatesynthesis by competitive inhibition of dihydropteroate synthetase,thereby inhibiting nucleic acid synthesis.

Tetracyclines include, but are not limited to, Demeclocycline,Doxycycline, Minocycline, Oxytetracycline, and Tetracycline.Tetracyclines are effective, e.g., against Gram-negative bacteria.Tetracyclines are believed to bind to the bacterial 30S ribosomalsubunit thereby inhibiting bacterial protein synthesis.

Anti-mycobacterial compounds include, but are not limited to,Clofazimine, Dapsone, Capreomycin, Cycloserine, Ethambutol, Ethionamide,Isoniazid, Pyrazinamide, Rifampicin, Rifabutin, Rifapentine, andStreptomycin.

Suitable antibiotics also include arsphenamine, chloramphenicol,fosfomycin, fusidic acid, metronidazole, mupirocin, platensimycin,quinupristin/dalfopristin, tigecycline, tinidazole, trimethoprimamoxicillin/clavulanate, ampicillin/sulbactam, amphomycin ristocetin,azithromycin, bacitracin, buforin II, carbomycin, cecropin Pl,clarithromycin, erythromycins, furazolidone, fusidic acid, Na fusidate,gramicidin, imipenem, indolicidin, josamycin, magainan II,metronidazole, nitroimidazoles, mikamycin, mutacin B-Ny266, mutacinB-JH1 140, mutacin J-T8, nisin, nisin A, novobiocin, oleandomycin,ostreogrycin, piperacillin/tazobactam, pristinamycin, ramoplanin,ranalexin, reuterin, rifaximin, rosamicin, rosaramicin, spectinomycin,spiramycin, staphylomycin, streptogramin, streptogramin A, synergistin,taurolidine, teicoplanin, telithromycin, ticarcillin/clavulanic acid,triacetyloleandomycin, tylosin, tyrocidin, tyrothricin, vancomycin,vemamycin, and virginiamycin.

In some embodiments, the additional therapeutic is an immunosuppressiveagent, a DMARD, a pain-control drug, a steroid, a non-steroidalantiinflammatory drug (NSAID), or a cytokine antagonist, andcombinations thereof. Representative agents include, but are not limitedto, cyclosporin, retinoids, corticosteroids, propionic acid derivative,acetic acid derivative, enolic acid derivatives, fenamic acidderivatives, Cox-2 inhibitors, lumiracoxib, ibuprophen, cholin magnesiumsalicylate, fenoprofen, salsalate, difunisal, tolmetin, ketoprofen,flurbiprofen, oxaprozin, indomethacin, sulindac, etodolac, ketorolac,nabumetone, naproxen, valdecoxib, etoricoxib, MK0966; rofecoxib,acetominophen, Celecoxib, Diclofenac, tramadol, piroxicam, meloxicam,tenoxicam, droxicam, lornoxicam, isoxicam, mefanamic acid, meclofenamicacid, flufenamic acid, tolfenamic, valdecoxib, parecoxib, etodolac,indomethacin, aspirin, ibuprophen, firocoxib, methotrexate (MTX),antimalarial drugs (e.g., hydroxychloroquine and chloroquine),sulfasalazine, Leflunomide, azathioprine, cyclosporin, gold salts,minocycline, cyclophosphamide, D-penicillamine, minocycline, auranofin,tacrolimus, myocrisin, chlorambucil, TNF alpha antagonists (e.g., TNFalpha antagonists or TNF alpha receptor antagonists), e.g., ADALIMUMAB(Humira®), ETANERCEPT (Enbrel®), INFLIXIMAB (Remicade®; TA-650),CERTOLIZUMAB PEGOL (Cimzia®; CDP870), GOLIMUMAB (Simpom®; CNTO 148),ANAKINRA (Kineret®), RITUXIMAB (Rituxan®; MabThera®), ABATACEPT(Orencia®), TOCILIZUMAB (RoActemra/Actemra®), integrin antagonists(TYSABRI® (natalizumab)), IL-1 antagonists (ACZ885 (Ilaris)), Anakinra(Kineret®)), CD4 antagonists, IL-23 antagonists, IL-20 antagonists, IL-6antagonists, BLyS antagonists (e.g., Atacicept, Benlysta®/LymphoStat-B®(belimumab)), p38 Inhibitors, CD20 antagonists (Ocrelizumab, Ofatumumab(Arzerra®)), interferon gamma antagonists (Fontolizumab), prednisolone,Prednisone, dexamethasone, Cortisol, cortisone, hydrocortisone,methylprednisolone, betamethasone, triamcinolone, beclometasome,fludrocortisone, deoxycorticosterone, aldosterone, Doxycycline,vancomycin, pioglitazone, SBI-087, SC10-469, Cura-100, Oncoxin+Viusid,TwHF, Methoxsalen, Vitamin D—ergocalciferol, Milnacipran, Paclitaxel,rosig tazone, Tacrolimus (Prograf®), RADOO1, rapamune, rapamycin,fostamatinib, Fentanyl, XOMA 052, Fostamatinib disodium, rosightazone,Curcumin (Longvida™) Rosuvastatin, Maraviroc, ramipnl, Milnacipran,Cobiprostone, somatropin, tgAAC94 gene therapy vector, MK0359, GW856553,esomeprazole, everolimus, trastuzumab, JAK1 and JAK2 inhibitors, pan JAKinhibitors, e.g., tetracyclic pyridone 6 (P6), 325, PF-956980,denosumab, IL-6 antagonists, CD20 antagonistis, CTLA4 antagonists, IL-8antagonists, IL-21 antagonists, IL-22 antagonist, integrin antagonists(Tysarbri® (natalizumab)), VGEF antagnosits, CXCL antagonists, MMPantagonists, defensin antagonists, IL-1 antagonists (including IL-1 betaantagonsits), and IL-23 antagonists (e.g., receptor decoys, antagonisticantibodies, etc.).

In some embodiments, the agent is an immunosuppressive agent. Examplesof immunosuppressive agents include, but are not limited to,corticosteroids, mesalazine, mesalamine, sulfasalazine, sulfasalazinederivatives, immunosuppressive drugs, cyclosporin A, mercaptopurine,azathiopurine, prednisone, methotrexate, antihistamines,glucocorticoids, epinephrine, theophylline, cromolyn sodium,anti-leukotrienes, anti-cholinergic drugs for rhinitis, TLR antagonists,inflammasome inhibitors, anti-cholinergic decongestants, mast-cellstabilizers, monoclonal anti-IgE antibodies, vaccines (e.g., vaccinesused for vaccination where the amount of an allergen is graduallyincreased), cytokine inhibitors, such as anti-IL-6 antibodies, TNFinhibitors such as infliximab, adalimumab, certolizumab pegol,golimumab, or etanercept, and combinations thereof.

In some embodiments, the immune disorder therapy comprises administeringa therapeutic bacteria and/or a therapeutic combination of bacteria tothe subject so a healthy microbiome can be reconstituted in the subject.In some embodiments, the therapeutic bacteria is anon-immune-disorder-associated bacteria. In some embodiments thetherapeutic bacteria is a probiotic bacteria.

In some embodiments, the additional therapeutic is a cancer therapeutic.In some embodiments, the cancer therapeutic is a chemotherapeutic agent.Examples of such chemotherapeutic agents include, but are not limitedto, alkylating agents such as cyclosphosphamide; alkyl sulfonates suchas busulfan, improsulfan and piposulfan; aziridines such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelaminesincluding altretamine, triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gammalI and calicheamicinomegal1; dynemicin, including dynemicin A; bisphosphonates, such asclodronate; an esperamicin; as well as neocarzinostatin chromophore andrelated chromoprotein enediyne antibiotic chromophores, aclacinomysins,actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin(including morpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharidecomplex); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonicacid; triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes(especially T-2 toxin, verracurin A, roridin A and anguidine); urethan;vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol;pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide;thiotepa; taxoids, e.g., paclitaxel and doxetaxel; chlorambucil;gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinumcoordination complexes such as cisplatin, oxaliplatin and carboplatin;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; vinorelbine; novantrone; teniposide; edatrexate;daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11);topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO);retinoids such as retinoic acid; capecitabine; and pharmaceuticallyacceptable salts, acids or derivatives of any of the above.

In some embodiments, the cancer therapeutic is a cancer immunotherapyagent. Immunotherapy refers to a treatment that uses a subject's immunesystem to treat cancer, e.g., checkpoint inhibitors, cancer vaccines,cytokines, cell therapy, CAR-T cells, and dendritic cell therapy.Non-limiting examples of immunotherapies are checkpoint inhibitorsinclude Nivolumab (BMS, anti-PD-1), Pembrolizumab (Merck, anti-PD-1),Ipilimumab (BMS, anti-CTLA-4), MEDI4736 (AstraZeneca, anti-PD-L1), andMPDL3280A (Roche, anti-PD-L1). Other immunotherapies may be tumorvaccines, such as Gardail, Cervarix, BCG, sipulencel-T, Gp100:209-217,AGS-003, DCVax-L, Algenpantucel-L, Tergenpantucel-L, TG4010, ProstAtak,Prostvac-V/R-TRICOM, Rindopepimul, E75 peptide acetate, IMA901,POL-103A, Belagenpumatucel-L, GSK1572932A, MDX-1279, GV1001, andTecemotide. Immunotherapy may be administered via injection (e.g.,intravenously, intratumorally, subcutaneously, or into lymph nodes), butmay also be administered orally, topically, or via aerosol.Immunotherapies may comprise adjuvants such as cytokines.

In some embodiments, the immunotherapy agent is an immune checkpointinhibitor. Immune checkpoint inhibition broadly refers to inhibiting thecheckpoints that cancer cells can produce to prevent or downregulate animmune response. Examples of immune checkpoint proteins include, but arenot limited to, CTLA4, PD-1, PD-L1, PD-L2, A2AR, B7-H3, B7-H4, BTLA,KIR, LAGS, TIM-3 or VISTA. Immune checkpoint inhibitors can beantibodies or antigen binding fragments thereof that bind to and inhibitan immune checkpoint protein. Examples of immune checkpoint inhibitorsinclude, but are not limited to, nivolumab, pembrolizumab, pidilizumab,AMP-224, AMP-514, STI-A1110, TSR-042, RG-7446, BMS-936559, MEDI-4736,MSB-0020718C, AUR-012 and STI-A1010.

In some embodiments, the immunotherapy agent is an antibody or antigenbinding fragment thereof that, for example, binds to a cancer-associatedantigen. Examples of cancer-associated antigens include, but are notlimited to, adipophilin, AIM-2, ALDH1A1, alpha-actinin-4,alpha-fetoprotein (“AFP”), ARTC1, B-RAF, BAGE-1, BCLX (L), BCR-ABLfusion protein b3a2, beta-catenin, BING-4, CA-125, CALCA,carcinoembryonic antigen (“CEA”), CASP-5, CASP-8, CD274, CD45, Cdc27,CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, CPSF, CSNK1A1, CTAG1, CTAG2,cyclin D1, Cyclin-A1, dek-can fusion protein, DKK1, EFTUD2, Elongationfactor 2, ENAH (hMena), Ep-CAM, EpCAM, EphA3, epithelial tumor antigen(“ETA”), ETV6-AML1 fusion protein, EZH2, FGF5, FLT3-ITD, FN1,G250/MN/CAIX, GAGE-1,2,8, GAGE-3,4,5,6,7, GAS7, glypican-3, GnTV,gp100/Pme117, GPNMB, HAUS3, Hepsin, HER-2/neu, HERV-K-MEL, HLA-A11,HLA-A2, HLA-DOB, hsp70-2, IDO1, IGF2B3, IL13Ralpha2, Intestinal carboxylesterase, K-ras, Kallikrein 4, KIF20A, KK-LC-1, KKLC1, KM-HN-1, KMHN1also known as CCDC110, LAGE-1, LDLR-fucosyltransferaseAS fusion protein,Lengsin, M-CSF, MAGE-A1, MAGE-A10, MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4,MAGE-A6, MAGE-A9, MAGE-C1, MAGE-C2, malic enzyme, mammaglobin-A, MART2,MATN, MC1R, MCSP, mdm-2, ME1, Melan-A/MART-1, Meloe, Midkine, MMP-2,MMP-7, MUC1, MUCSAC, mucin, MUM-1, MUM-2, MUM-3, Myosin, Myosin class I,N-raw, NA88-A, neo-PAP, NFYC, NY-BR-1, NY-ESO-1/LAGE-2, OA1, OGT, OS-9,P polypeptide, p53, PAP, PAX5, PBF, pml-RARalpha fusion protein,polymorphic epithelial mucin (“PEM”), PPP1R3B, PRAME, PRDX5, PSA, PSMA,PTPRK, RAB38/NY-MEL-1, RAGE-1, RBAF600, RGS5, RhoC, RNF43, RU2AS, SAGE,secernin 1, SIRT2, SNRPD1, SOX10, Sp17, SPA17, SSX-2, SSX-4, STEAP1,survivin, SYT-SSX1 or -SSX2 fusion protein, TAG-1, TAG-2, Telomerase,TGF-betaRll, TPBG, TRAG-3, Triosephosphate isomerase, TRP-1/gp75, TRP-2,TRP2-INT2, tyrosinase, tyrosinase (“TYR”), VEGF, WT1, XAGE-1b/GAGED2a.In some embodiments, the antigen is a neo-antigen.

In some embodiments, the immunotherapy agent is a cancer vaccine and/ora component of a cancer vaccine (e.g., an antigenic peptide and/orprotein). The cancer vaccine can be a protein vaccine, a nucleic acidvaccine or a combination thereof. For example, in some embodiments, thecancer vaccine comprises a polypeptide comprising an epitope of acancer-associated antigen. In some embodiments, the cancer vaccinecomprises a nucleic acid (e.g., DNA or RNA, such as mRNA) that encodesan epitope of a cancer-associated antigen. In some embodiments, thenucleic acid is a vector (e.g., a bacterial vector, viral vector).Examples of bacterial vectors include, but are not limited to,Mycobacterium bovis (BCG), Salmonella Typhimurium ssp., Salmonella Typhissp., Clostridium sp. spores, Escherichia coli Nissle 1917, Escherichiacoli K-12/LLO, Listeria monocytogenes, and Shigella flexneri. Examplesof viral vectors include, but are not limited to, vaccinia, adenovirus,RNA viruses, and replication defective avipox, replication-defectivefowlpox, replication-defective canarypox, replication defective MVA andreplication-defective adenovirus.

In some embodiments, the cancer immunotherapy comprises administrationof an antigen presenting cell (APC) primed with a cancer-specificantigen. In some embodiments, the APC is a dendritic cell, a macrophageor a B cell.

Examples of cancer-associated antigens include, but are not limited to,adipophilin, AIM-2, ALDH1A1, alpha-actinin-4, alpha-fetoprotein (“AFP”),ARTC1, B-RAF, BAGE-1, BCLX (L), BCR-ABL fusion protein b3a2,beta-catenin, BING-4, CA-125, CALCA, carcinoembryonic antigen (“CEA”),CASP-5, CASP-8, CD274, CD45, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLPP,COA-1, CPSF, CSNK1A1, CTAG1, CTAG2, cyclin D1, Cyclin-A1, dek-can fusionprotein, DKK1, EFTUD2, Elongation factor 2, ENAH (hMena), Ep-CAM, EpCAM,EphA3, epithelial tumor antigen (“ETA”), ETV6-AML1 fusion protein, EZH2,FGF5, FLT3-ITD, FN1, G250/MN/CAIX, GAGE-1,2,8, GAGE-3,4,5,6,7, GAS7,glypican-3, GnTV, gp100/Pme117, GPNMB, HAUS3, Hepsin, HER-2/neu,HERV-K-MEL, HLA-A11, HLA-A2, HLA-DOB, hsp70-2, IDO1, IGF2B3,IL13Ralpha2, Intestinal carboxyl esterase, K-ras, Kallikrein 4, KIF20A,KK-LC-1, KKLC1, KM-HN-1, KMHN1 also known as CCDC110, LAGE-1,LDLR-fucosyltransferaseAS fusion protein, Lengsin, M-CSF, MAGE-A1,MAGE-A10, MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9,MAGE-C1, MAGE-C2, malic enzyme, mammaglobin-A, MART2, MATN, MC1R, MCSP,mdm-2, ME1, Melan-A/MART-1, Meloe, Midkine, MMP-2, MMP-7, MUC1, MUC5AC,mucin, MUM-1, MUM-2, MUM-3, Myosin, Myosin class I, N-raw, NA88-A,neo-PAP, NFYC, NY-BR-1, NY-ESO-1/LAGE-2, OA1, OGT, OS-9, P polypeptide,p53, PAP, PAX5, PBF, pml-RARalpha fusion protein, polymorphic epithelialmucin (“PEM”), PPP1R3B, PRAME, PRDX5, PSA, PSMA, PTPRK, RAB38/NY-MEL-1,RAGE-1, RBAF600, RGS5, RhoC, RNF43, RU2AS, SAGE, secernin 1, SIRT2,SNRPD1, SOX10, Sp17, SPA17, SSX-2, SSX-4, STEAP1, survivin, SYT-SSX1 or-SSX2 fusion protein, TAG-1, TAG-2, Telomerase, TGF-betaRll, TPBG,TRAG-3, Triosephosphate isomerase, TRP-1/gp75, TRP-2, TRP2-INT2,tyrosinase, tyrosinase (“TYR”), VEGF, WT1, XAGE-1b/GAGED2a. In someembodiments, the antigen is a neo-antigen.

In some embodiments, the cancer immunotherapy comprises administrationof a cancer-specific chimeric antigen receptor (CAR). In someembodiments, the CAR is administered on the surface of a T cell. In someembodiments, the CAR binds specifically to a cancer-associated antigen.

In some embodiments, the cancer immunotherapy comprises administrationof a cancer-specific T cell to the subject. In some embodiments, the Tcell is a CD4+ T cell. In some embodiments, the CD4+ T cell is a TH1 Tcell, a TH2 T cell or a TH17 T cell. In some embodiments, the T cellexpresses a T cell receptor specific for a cancer-associated antigen.

In some embodiments, the cancer vaccine is administered with anadjuvant. Examples of adjuvants include, but are not limited to, animmune modulatory protein, Adjuvant 65, α-GalCer, aluminum phosphate,aluminum hydroxide, calcium phosphate, β-Glucan Peptide, CpG ODN DNA,GPI-0100, lipid A, lipopolysaccharide, Lipovant, Montanide,N-acetyl-muramyl-L-alanyl-D-isoglutamine, Pam3CSK4, quil A, choleratoxin (CT) and heat-labile toxin from enterotoxigenic Escherichia coli(LT) including derivatives of these (CTB, mmCT, CTA1-DD, LTB, LTK63,LTR72, dmLT) and trehalose dimycolate.

In some embodiments, the immunotherapy agent is an immune modulatingprotein to the subject. In some embodiments, the immune modulatoryprotein is a cytokine or chemokine. Examples of immune modulatingproteins include, but are not limited to, B lymphocyte chemoattractant(“BLC”), C—C motif chemokine 11 (“Eotaxin-1”), Eosinophil chemotacticprotein 2 (“Eotaxin-2”), Granulocyte colony-stimulating factor(“G-CSF”), Granulocyte macrophage colony-stimulating factor (“GM-CSF”),1-309, Intercellular Adhesion Molecule 1 (“ICAM-1”), Interferon alpha(“IFN-alpha”), Interferon beta (“IFN-beta”) Interferon gamma(“IFN-gamma”), Interlukin-1 alpha (“IL-1 alpha”), Interlukin-1 beta(“IL-1 beta”), Interleukin 1 receptor antagonist (“IL-1 ra”),Interleukin-2 (“IL-2”), Interleukin-4 (“IL-4”), Interleukin-5 (“IL-5”),Interleukin-6 (“IL-6”), Interleukin-6 soluble receptor (“IL-6 sR”),Interleukin-7 (“IL-7”), Interleukin-8 (“IL-8”), Interleukin-10(“IL-10”), Interleukin-11 (“IL-11”), Subunit beta of Interleukin-12(“IL-12 p40” or “IL-12 p70”), Interleukin-13 (“IL-13”), Interleukin-15(“IL-15”), Interleukin-16 (“IL-16”), Interleukin-17A-F (“IL-17A-F”),Interleukin-18 (“IL-18”), Interleukin-21 (“IL-21”), Interleukin-22(“IL-22”), Interleukin-23 (“IL-23”), Interleukin-33 (“IL-33”), Chemokine(C—C motif) Ligand 2 (“MCP-1”), Macrophage colony-stimulating factor(“M-CSF”), Monokine induced by gamma interferon (“MIG”), Chemokine (C—Cmotif) ligand 2 (“MIP-1 alpha”), Chemokine (C—C motif) ligand 4 (“MIP-1beta”), Macrophage inflammatory protein-1-delta (“MIP-1 delta”),Platelet-derived growth factor subunit B (“PDGF-BB”), Chemokine (C—Cmotif) ligand 5, Regulated on Activation, Normal T cell Expressed andSecreted (“RANTES”), TIMP metallopeptidase inhibitor 1 (“TIMP-1”), TIMPmetallopeptidase inhibitor 2 (“TIMP-2”), Tumor necrosis factor,lymphotoxin-alpha (“TNF alpha”), Tumor necrosis factor, lymphotoxin-beta(“TNF beta”), Soluble TNF receptor type 1 (“sTNFRI”), sTNFRIIAR,Brain-derived neurotrophic factor (“BDNF”), Basic fibroblast growthfactor (“bFGF”), Bone morphogenetic protein 4 (“BMP-4”), Bonemorphogenetic protein 5 (“BMP-5”), Bone morphogenetic protein 7(“BMP-7”), Nerve growth factor (“b-NGF”), Epidermal growth factor(“EGF”), Epidermal growth factor receptor (“EGFR”),Endocrine-gland-derived vascular endothelial growth factor (“EG-VEGF”),Fibroblast growth factor 4 (“FGF-4”), Keratinocyte growth factor(“FGF-7”), Growth differentiation factor 15 (“GDF-15”), Glialcell-derived neurotrophic factor (“GDNF”), Growth Hormone,Heparin-binding EGF-like growth factor (“HB-EGF”), Hepatocyte growthfactor (“HGF”), Insulin-like growth factor binding protein 1(“IGFBP-1”), Insulin-like growth factor binding protein 2 (“IGFBP-2”),Insulin-like growth factor binding protein 3 (“IGFBP-3”), Insulin-likegrowth factor binding protein 4 (“IGFBP-4”), Insulin-like growth factorbinding protein 6 (“IGFBP-6”), Insulin-like growth factor 1 (“IGF-1”),Insulin, Macrophage colony-stimulating factor (“M-CSF R”), Nerve growthfactor receptor (“NGF R”), Neurotrophin-3 (“NT-3”), Neurotrophin-4(“NT-4”), Osteoclastogenesis inhibitory factor (“Osteoprotegerin”),Platelet-derived growth factor receptors (“PDGF-AA”),Phosphatidylinositol-glycan biosynthesis (“PIGF”), Skp, Cullin, F-boxcontaining comples (“SCF”), Stem cell factor receptor (“SCF R”),Transforming growth factor alpha (“TGFalpha”), Transforming growthfactor beta-1 (“TGF beta 1”), Transforming growth factor beta-3 (“TGFbeta 3”), Vascular endothelial growth factor (“VEGF”), Vascularendothelial growth factor receptor 2 (“VEGFR2”), Vascular endothelialgrowth factor receptor 3 (“VEGFR3”), VEGF-D 6Ckine, Tyrosine-proteinkinase receptor UFO (“Axl”), Betacellulin (“BTC”), Mucosae-associatedepithelial chemokine (“CCL28”), Chemokine (C—C motif) ligand 27(“CTACK”), Chemokine (C—X—C motif) ligand 16 (“CXCL16”), C—X—C motifchemokine 5 (“ENA-78”), Chemokine (C—C motif) ligand 26 (“Eotaxin-3”),Granulocyte chemotactic protein 2 (“GCP-2”), GRO, Chemokine (C—C motif)ligand 14 (“HCC-1”), Chemokine (C—C motif) ligand 16 (“HCC-4”),Interleukin-9 (“IL-9”), Interleukin-17 F (“IL-17F”),Interleukin-18-binding protein (“IL-18 BPa”), Interleukin-28 A(“IL-28A”), Interleukin 29 (“IL-29”), Interleukin 31 (“IL-31”), C—X—Cmotif chemokine 10 (“IP-10”), Chemokine receptor CXCR3 (“I-TAC”),Leukemia inhibitory factor (“LIF”), Light, Chemokine (C motif) ligand(“Lymphotactin”), Monocyte chemoattractant protein 2 (“MCP-2”), Monocytechemoattractant protein 3 (“MCP-3”), Monocyte chemoattractant protein 4(“MCP-4”), Macrophage-derived chemokine (“MDC”), Macrophage migrationinhibitory factor (“MIF”), Chemokine (C—C motif) ligand 20 (“MIP-3alpha”), C—C motif chemokine 19 (“MIP-3 beta”), Chemokine (C—C motif)ligand 23 (“MPIF-1”), Macrophage stimulating protein alpha chain(“MSPalpha”), Nucleosome assembly protein 1-like 4 (“NAP-2”), Secretedphosphoprotein 1 (“Osteopontin”), Pulmonary and activation-regulatedcytokine (“PARC”), Platelet factor 4 (“PF4”), Stroma cell-derivedfactor-1 alpha (“SDF-1 alpha”), Chemokine (C—C motif) ligand 17(“TARC”), Thymus-expressed chemokine (“TECK”), Thymic stromallymphopoietin (“TSLP 4-IBB”), CD 166 antigen (“ALCAM”), Cluster ofDifferentiation 80 (“B7-1”), Tumor necrosis factor receptor superfamilymember 17 (“BCMA”), Cluster of Differentiation 14 (“CD14”), Cluster ofDifferentiation 30 (“CD30”), Cluster of Differentiation 40 (“CD40Ligand”), Carcinoembryonic antigen-related cell adhesion molecule 1(biliary glycoprotein) (“CEACAM-1”), Death Receptor 6 (“DR6”),Deoxythymidine kinase (“Dtk”), Type 1 membrane glycoprotein(“Endoglin”), Receptor tyrosine-protein kinase erbB-3 (“ErbB3”),Endothelial-leukocyte adhesion molecule 1 (“E-Selectin”), Apoptosisantigen 1 (“Fas”), Fms-like tyrosine kinase 3 (“Flt-3L”), Tumor necrosisfactor receptor superfamily member 1 (“GITR”), Tumor necrosis factorreceptor superfamily member 14 (“HVEM”), Intercellular adhesion molecule3 (“ICAM-3”), IL-1 R4, IL-1 RI, IL-10 Rbeta, IL-17R, IL-2Rgamma, IL-21R,Lysosome membrane protein 2 (“LIMPII”), Neutrophil gelatinase-associatedlipocalin (“Lipocalin-2”), CD62L (“L-Selectin”), Lymphatic endothelium(“LYVE-1”), MHC class I polypeptide-related sequence A (“MICA”), MHCclass I polypeptide-related sequence B (“MICB”), NRG1-betal, Beta-typeplatelet-derived growth factor receptor (“PDGF Rbeta”), Plateletendothelial cell adhesion molecule (“PECAM-1”), RAGE, Hepatitis A viruscellular receptor 1 (“TIM-1”), Tumor necrosis factor receptorsuperfamily member IOC (“TRAIL R3”), Trappin protein transglutaminasebinding domain (“Trappin-2”), Urokinase receptor (“uPAR”), Vascular celladhesion protein 1 (“VCAM-1”), XEDARActivin A, Agouti-related protein(“AgRP”), Ribonuclease 5 (“Angiogenin”), Angiopoietin 1, Angiostatin,Catheprin S, CD40, Cryptic family protein IB (“Cripto-1”), DAN,Dickkopf-related protein 1 (“DKK-1”), E-Cadherin, Epithelial celladhesion molecule (“EpCAM”), Fas Ligand (FasL or CD95L), Fcg RIIB/C,FoUistatin, Galectin-7, Intercellular adhesion molecule 2 (“ICAM-2”),IL-13 R1, IL-13R2, IL-17B, IL-2 Ra, IL-2 Rb, IL-23, LAP, Neuronal celladhesion molecule (“NrCAM”), Plasminogen activator inhibitor-1(“PAI-1”), Platelet derived growth factor receptors (“PDGF-AB”),Resistin, stromal cell-derived factor 1 (“SDF-1 beta”), sgp130, Secretedfrizzled-related protein 2 (“ShhN”), Sialic acid-bindingimmunoglobulin-type lectins (“Siglec-5”), ST2, Transforming growthfactor-beta 2 (“TGF beta 2”), Tie-2, Thrombopoietin (“TPO”), Tumornecrosis factor receptor superfamily member 10D (“TRAIL R4”), Triggeringreceptor expressed on myeloid cells 1 (“TREM-1”), Vascular endothelialgrowth factor C (“VEGF-C”), VEGFRlAdiponectin, Adipsin (“AND”),Alpha-fetoprotein (“AFP”), Angiopoietin-like 4 (“ANGPTL4”),Beta-2-microglobulin (“B2M”), Basal cell adhesion molecule (“BCAM”),Carbohydrate antigen 125 (“CA125”), Cancer Antigen 15-3 (“CA15-3”),Carcinoembryonic antigen (“CEA”), cAMP receptor protein (“CRP”), HumanEpidermal Growth Factor Receptor 2 (“ErbB2”), Follistatin,Follicle-stimulating hormone (“FSH”), Chemokine (C—X—C motif) ligand 1(“GRO alpha”), human chorionic gonadotropin (“beta HCG”), Insulin-likegrowth factor 1 receptor (“IGF-1 sR”), IL-1 sRII, IL-3, IL-18 Rb, IL-21,Leptin, Matrix metalloproteinase-1 (“MMP-1”), Matrix metalloproteinase-2(“MMP-2”), Matrix metalloproteinase-3 (“MMP-3”), Matrixmetalloproteinase-8 (“MMP-8”), Matrix metalloproteinase-9 (“MMP-9”),Matrix metalloproteinase-10 (“MMP-10”), Matrix metalloproteinase-13(“MMP-13”), Neural Cell Adhesion Molecule (“NCAM-1”), Entactin(“Nidogen-1”), Neuron specific enolase (“NSE”), Oncostatin M (“OSM”),Procalcitonin, Prolactin, Prostate specific antigen (“PSA”), Sialicacid-binding Ig-like lectin 9 (“Siglec-9”), ADAM 17 endopeptidase(“TACE”), Thyroglobulin, Metalloproteinase inhibitor 4 (“TIMP-4”),TSH2B4, Disintegrin and metalloproteinase domain-containing protein 9(“ADAM-9”), Angiopoietin 2, Tumor necrosis factor ligand superfamilymember 13/Acidic leucine-rich nuclear phosphoprotein 32 family member B(“APRIL”), Bone morphogenetic protein 2 (“BMP-2”), Bone morphogeneticprotein 9 (“BMP-9”), Complement component 5a (“C5a”), Cathepsin L,CD200, CD97, Chemerin, Tumor necrosis factor receptor superfamily member6B (“DcR3”), Fatty acid-binding protein 2 (“FABP2”), Fibroblastactivation protein, alpha (“FAP”), Fibroblast growth factor 19(“FGF-19”), Galectin-3, Hepatocyte growth factor receptor (“HGF R”),IFN-gammalpha/beta R2, Insulin-like growth factor 2 (“IGF-2”),Insulin-like growth factor 2 receptor (“IGF-2 R”), Interleukin-1receptor 6 (“IL-1R6”), Interleukin 24 (“IL-24”), Interleukin 33(“IL-33”, Kallikrein 14, Asparaginyl endopeptidase (“Legumain”),Oxidized low-density lipoprotein receptor 1 (“LOX-1”), Mannose-bindinglectin (“MBL”), Neprilysin (“NEP”), Notch homolog 1,translocation-associated (Drosophila) (“Notch-1”), Nephroblastomaoverexpressed (“NOV”), Osteoactivin, Programmed cell death protein 1(“PD-1”), N-acetylmuramoyl-L-alanine amidase (“PGRP-5”), Serpin A4,Secreted frizzled related protein 3 (“sFRP-3”), Thrombomodulin, Tolllikereceptor 2 (“TLR2”), Tumor necrosis factor receptor superfamily member10A (“TRAIL R1”), Transferrin (“TRF”), WIF-1ACE-2, Albumin, AMICA,Angiopoietin 4, B-cell activating factor (“BAFF”), Carbohydrate antigen19-9 (“CA19-9”), CD 163, Clusterin, CRT AM, Chemokine (C—X—C motif)ligand 14 (“CXCL14”), Cystatin C, Decorin (“DCN”), Dickkopf-relatedprotein 3 (“Dkk-3”), Delta-like protein 1 (“DLL1”), Fetuin A,Heparin-binding growth factor 1 (“aFGF”), Folate receptor alpha(“FOLR1”), Furin, GPCR-associated sorting protein 1 (“GASP-1”),GPCR-associated sorting protein 2 (“GASP-2”), Granulocytecolony-stimulating factor receptor (“GCSF R”), Serine protease hepsin(“HAI-2”), Interleukin-17B Receptor (“IL-17B R”), Interleukin 27(“IL-27”), Lymphocyte-activation gene 3 (“LAG-3”), Apolipoprotein A-V(“LDL R”), Pepsinogen I, Retinol binding protein 4 (“RBP4”), SOST,Heparan sulfate proteoglycan (“Syndecan-1”), Tumor necrosis factorreceptor superfamily member 13B (“TACI”), Tissue factor pathwayinhibitor (“TFPI”), TSP-1, Tumor necrosis factor receptor superfamily,member 10b (“TRAIL R2”), TRANCE, Troponin I, Urokinase PlasminogenActivator (“uPA”), Cadherin 5, type 2 or VE-cadherin (vascularendothelial) also known as CD144 (“VE-Cadherin”),WNT1-inducible-signaling pathway protein 1 (“WISP-1”), and ReceptorActivator of Nuclear Factor κ B (“RANK”).

In some embodiments, the cancer therapeutic agent is an anti-cancercompound. Exemplary anti-cancer compounds include, but are not limitedto, Alemtuzumab (Campath®), Alitretinoin (Panretin®), Anastrozole(Arimidex®), Bevacizumab (Avastin®), Bexarotene (Targretin®), Bortezomib(Velcade®), Bosutinib (Bosulif®), Brentuximab vedotin (Adcetris®),Cabozantinib (Cometriq™), Carfilzomib (Kyprolis™), Cetuximab (Erbitux®),Crizotinib (Xalkori®), Dasatinib (Sprycel®), Denileukin diftitox(Ontak®), Erlotinib hydrochloride (Tarceva®), Everolimus (Afinitor®),Exemestane (Aromasin®), Fulvestrant (Faslodex®), Gefitinib (Iressa®),Ibritumomab tiuxetan (Zevalin®), Imatinib mesylate (Gleevec®),Ipilimumab (Yervoy™), Lapatinib ditosylate (Tykerb®), Letrozole(Femara®), Nilotinib (Tasigna®), Ofatumumab (Arzerra®), Panitumumab(Vectibix®), Pazopanib hydrochloride (Votrient®), Pertuzumab (Perjeta™),Pralatrexate (Folotyn®), Regorafenib (Stivarga®), Rituximab (Rituxan®),Romidepsin (Istodax®), Sorafenib tosylate (Nexavar®), Sunitinib malate(Sutent®), Tamoxifen, Temsirolimus (Torisel®), Toremifene (Fareston®),Tositumomab and 131I-tositumomab (Bexxar®), Trastuzumab (Herceptin®),Tretinoin (Vesanoid®), Vandetanib (Caprelsa®), Vemurafenib (Zelboraf®),Vorinostat (Zolinza®), and Ziv-aflibercept (Zaltrap®).

Exemplary anti-cancer compounds that modify the function of proteinsthat regulate gene expression and other cellular functions (e.g., HDACinhibitors, retinoid receptor ligants) are Vorinostat (Zolinza®),Bexarotene (Targretin®) and Romidepsin (Istodax®), Alitretinoin(Panretin®), and Tretinoin (Vesanoid®).

Exemplary anti-cancer compounds that induce apoptosis (e.g., proteasomeinhibitors, antifolates) are Bortezomib (Velcade®), Carfilzomib(Kyprolis™), and Pralatrexate (Folotyn®).

Exemplary anti-cancer compounds that increase anti-tumor immune response(e.g., anti CD20, anti CD52; anti-cytotoxic T-lymphocyte-associatedantigen-4) are Rituximab (Rituxan®), Alemtuzumab (Campath®), Ofatumumab(Arzerra®), and Ipilimumab (Yervoy™)

Exemplary anti-cancer compounds that deliver toxic agents to cancercells (e.g., anti-CD20-radionuclide fusions; IL-2-diphtheria toxinfusions; anti-CD30-monomethylauristatin E (MMAE)-fusions) areTositumomab and 131I-tositumomab (Bexxar®) and Ibritumomab tiuxetan(Zevalin®), Denileukin diftitox (Ontak®), and Brentuximab vedotin(Adcetris®).

Other exemplary anti-cancer compounds are small molecule inhibitors andconjugates thereof of, e.g., Janus kinase, ALK, Bcl-2, PARP, PI3K, VEGFreceptor, Braf, MEK, CDK, and HSP90.

Exemplary platinum-based anti-cancer compounds include, for example,cisplatin, carboplatin, oxaliplatin, satraplatin, picoplatin,Nedaplatin, Triplatin, and Lipoplatin. Other metal-based drugs suitablefor treatment include, but are not limited to ruthenium-based compounds,ferrocene derivatives, titanium-based compounds, and gallium-basedcompounds.

In some embodiments, the cancer therapeutic is a radioactive moiety thatcomprises a radionuclide. Exemplary radionuclides include, but are notlimited to Cr-51, Cs-131, Ce-134, Se-75, Ru-97, 1-125, Eu-149, Os-189m,Sb-119, 1-123, Ho-161, Sb-117, Ce-139, In-111, Rh-103m, Ga-67, T1-201,Pd-103, Au-195, Hg-197, Sr-87m, Pt-191, P-33, Er-169, Ru-103, Yb-169,Au-199, Sn-121, Tm-167, Yb-175, In-113m, Sn-113, Lu-177, Rh-105,Sn-117m, Cu-67, Sc-47, Pt-195m, Ce-141, 1-131, Tb-161, As-77, Pt-197,Sm-153, Gd-159, Tm-173, Pr-143, Au-198, Tm-170, Re-186, Ag-111, Pd-109,Ga-73, Dy-165, Pm-149, Sn-123, Sr-89, Ho-166, P-32, Re-188, Pr-142,Ir-194, In-114m/In-114, and Y-90.

Immune Disorders

In some embodiments, the methods and compositions described hereinrelate to the treatment or prevention of a disease or disorderassociated with a pathological immune response, such as an autoimmunedisease, an allergic reaction and/or an inflammatory disease. In someembodiments, the disease or disorder is an inflammatory bowel disease(e.g., Crohn's disease or ulcerative colitis). In some embodiments, themethods and compositions described herein relate to the treatment orprevention of delayed-type hypersensitivity, autoimmune myocarditis,granulomas, peripheral neuropathies, Hashimoto's thyroiditis,inflammation of the colon, colitis, microscopic colitis, collagenouscolitis, diversion colitis, chemical colitis, ischemic colitis,indeterminate colitis, atypical colitis.

The methods described herein can be used to treat any subject in needthereof. As used herein, a “subject in need thereof” includes anysubject that has a disease or disorder associated with a pathologicalimmune response (e.g., an inflammatory bowel disease), as well as anysubject with an increased likelihood of acquiring a such a disease ordisorder.

The compositions described herein can be used, for example, as apharmaceutical composition for preventing or treating (reducing,partially or completely, the adverse effects of) an autoimmune disease,such as chronic inflammatory bowel disease, systemic lupuserythematosus, psoriasis, muckle-wells syndrome, rheumatoid arthritis,multiple sclerosis, or Hashimoto's disease; an allergic disease, such asa food allergy, pollenosis, or asthma; an infectious disease, such as aninfection with Clostridium difficile; an inflammatory disease such as aTNF-mediated inflammatory disease (e.g., an inflammatory disease of thegastrointestinal tract, such as pouchitis, a cardiovascular inflammatorycondition, such as atherosclerosis, or an inflammatory lung disease,such as chronic obstructive pulmonary disease); a pharmaceuticalcomposition for suppressing rejection in organ transplantation or othersituations in which tissue rejection might occur; a supplement, food, orbeverage for improving immune functions; or a reagent for suppressingthe proliferation or function of immune cells.

In some embodiments, the methods provided herein are useful for thetreatment of inflammation. In certain embodiments, the inflammation ofany tissue and organs of the body, including musculoskeletalinflammation, vascular inflammation, neural inflammation, digestivesystem inflammation, ocular inflammation, inflammation of thereproductive system, and other inflammation, as discussed below.

Immune disorders of the musculoskeletal system include, but are notlimited, to those conditions affecting skeletal joints, including jointsof the hand, wrist, elbow, shoulder, jaw, spine, neck, hip, knew, ankle,and foot, and conditions affecting tissues connecting muscles to bonessuch as tendons. Examples of such immune disorders, which may be treatedwith the methods and compositions described herein include, but are notlimited to, arthritis (including, for example, osteoarthritis,rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, acuteand chronic infectious arthritis, arthritis associated with gout andpseudogout, and juvenile idiopathic arthritis), tendonitis, synovitis,tenosynovitis, bursitis, fibrositis (fibromyalgia), epicondylitis,myositis, and osteitis (including, for example, Paget's disease,osteitis pubis, and osteitis fibrosa cystic).

Ocular immune disorders refers to an immune disorder that affects anystructure of the eye, including the eye lids. Examples of ocular immunedisorders which may be treated with the methods and compositionsdescribed herein include, but are not limited to, blepharitis,blepharochalasis, conjunctivitis, dacryoadenitis, keratitis,keratoconjunctivitis sicca (dry eye), scleritis, trichiasis, and uveitis

Examples of nervous system immune disorders which may be treated withthe methods and compositions described herein include, but are notlimited to, encephalitis, Guillain-Barre syndrome, meningitis,neuromyotonia, narcolepsy, multiple sclerosis, myelitis andschizophrenia. Examples of inflammation of the vasculature or lymphaticsystem which may be treated with the methods and compositions describedherein include, but are not limited to, arthrosclerosis, arthritis,phlebitis, vasculitis, and lymphangitis.

Examples of digestive system immune disorders which may be treated withthe methods and compositions described herein include, but are notlimited to, cholangitis, cholecystitis, enteritis, enterocolitis,gastritis, gastroenteritis, inflammatory bowel disease, ileitis, andproctitis. Inflammatory bowel diseases include, for example, certainart-recognized forms of a group of related conditions. Several majorforms of inflammatory bowel diseases are known, with Crohn's disease(regional bowel disease, e.g., inactive and active forms) and ulcerativecolitis (e.g., inactive and active forms) the most common of thesedisorders. In addition, the inflammatory bowel disease encompassesirritable bowel syndrome, microscopic colitis, lymphocytic-plasmocyticenteritis, coeliac disease, collagenous colitis, lymphocytic colitis andeosinophilic enterocolitis. Other less common forms of IBD includeindeterminate colitis, pseudomembranous colitis (necrotizing colitis),ischemic inflammatory bowel disease, Behcet's disease, sarcoidosis,scleroderma, IBD-associated dysplasia, dysplasia associated masses orlesions, and primary sclerosing cholangitis.

Examples of reproductive system immune disorders which may be treatedwith the methods and compositions described herein include, but are notlimited to, cervicitis, chorioamnionitis, endometritis, epididymitis,omphalitis, oophoritis, orchitis, salpingitis, tubo-ovarian abscess,urethritis, vaginitis, vulvitis, and vulvodynia.

The methods and compositions described herein may be used to treatautoimmune conditions having an inflammatory component. Such conditionsinclude, but are not limited to, acute disseminated alopeciauniversalise, Behcet's disease, Chagas' disease, chronic fatiguesyndrome, dysautonomia, encephalomyelitis, ankylosing spondylitis,aplastic anemia, hidradenitis suppurativa, autoimmune hepatitis,autoimmune oophoritis, celiac disease, Crohn's disease, diabetesmellitus type 1, giant cell arteritis, goodpasture's syndrome, Grave'sdisease, Guillain-Barre syndrome, Hashimoto's disease, Henoch-Schonleinpurpura, Kawasaki's disease, lupus erythematosus, microscopic colitis,microscopic polyarteritis, mixed connective tissue disease, Muckle-Wellssyndrome, multiple sclerosis, myasthenia gravis, opsoclonus myoclonussyndrome, optic neuritis, ord's thyroiditis, pemphigus, polyarteritisnodosa, polymyalgia, rheumatoid arthritis, Reiter's syndrome, Sjogren'ssyndrome, temporal arteritis, Wegener's granulomatosis, warm autoimmunehaemolytic anemia, interstitial cystitis, Lyme disease, morphea,psoriasis, sarcoidosis, scleroderma, ulcerative colitis, and vitiligo.

The methods and compositions described herein may be used to treatT-cell mediated hypersensitivity diseases having an inflammatorycomponent. Such conditions include, but are not limited to, contacthypersensitivity, contact dermatitis (including that due to poison ivy),uticaria, skin allergies, respiratory allergies (hay fever, allergicrhinitis, house dustmite allergy) and gluten-sensitive enteropathy(Celiac disease).

Other immune disorders which may be treated with the methods andcompositions include, for example, appendicitis, dermatitis,dermatomyositis, endocarditis, fibrositis, gingivitis, glossitis,hepatitis, hidradenitis suppurativa, iritis, laryngitis, mastitis,myocarditis, nephritis, otitis, pancreatitis, parotitis, percarditis,peritonoitis, pharyngitis, pleuritis, pneumonitis, prostatistis,pyelonephritis, and stomatisi, transplant rejection (involving organssuch as kidney, liver, heart, lung, pancreas (e.g., islet cells), bonemarrow, cornea, small bowel, skin allografts, skin homografts, and heartvalve xengrafts, sewrum sickness, and graft vs host disease), acutepancreatitis, chronic pancreatitis, acute respiratory distress syndrome,Sexary's syndrome, congenital adrenal hyperplasis, nonsuppurativethyroiditis, hypercalcemia associated with cancer, pemphigus, bullousdermatitis herpetiformis, severe erythema multiforme, exfoliativedermatitis, seborrheic dermatitis, seasonal or perennial allergicrhinitis, bronchial asthma, contact dermatitis, atopic dermatitis, drughypersensistivity reactions, allergic conjunctivitis, keratitis, herpeszoster ophthalmicus, iritis and oiridocyclitis, chorioretinitis, opticneuritis, symptomatic sarcoidosis, fulminating or disseminated pulmonarytuberculosis chemotherapy, idiopathic thrombocytopenic purpura inadults, secondary thrombocytopenia in adults, acquired (autoimmune)haemolytic anemia, leukaemia and lymphomas in adults, acute leukaemia ofchildhood, regional enteritis, autoimmune vasculitis, multiplesclerosis, chronic obstructive pulmonary disease, solid organ transplantrejection, sepsis. Preferred treatments include treatment of transplantrejection, rheumatoid arthritis, psoriatic arthritis, multiplesclerosis, Type 1 diabetes, asthma, inflammatory bowel disease, systemiclupus erythematosus, psoriasis, chronic obstructive pulmonary disease,and inflammation accompanying infectious conditions (e.g., sepsis).

The methods and compositions described herein may be used to treatmetabolic disorders and metabolic syndromes. Such conditions include,but are not limited to, Type II Diabetes, Encephalopathy, Tay-Sachsdisease, Krabbe disease, Galactosemia, Phenylketonuria (PKU), and Maplesyrup urine disease.

The methods and compositions described herein may be used to treatneurodegenerative and neurological diseases. Such conditions include,but are not limited to, Parkinson's disease, Alzheimer's disease, priondisease, Huntington's disease, motor neurone diseases (MND),spinocerebellar ataxia, spinal muscular atrophy, dystonia, idiopathicintracranial hypertension, epilepsy, nervous system disease, centralnervous system disease, movement disorders, multiple sclerosis,encephalopathy, and, post-operative cognitive dysfunction.

Cancer

In some embodiments, the methods and compositions described hereinrelate to the treatment of cancer. In some embodiments, any cancer canbe treated using the methods described herein. Examples of cancers thatmay treated by methods and compositions described herein include, butare not limited to, cancer cells from the bladder, blood, bone, bonemarrow, brain, breast, colon, esophagus, gastrointestine, gum, head,kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach,testis, tongue, or uterus. In addition, the cancer may specifically beof the following histological type, though it is not limited to these:neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant andspindle cell carcinoma; small cell carcinoma; papillary carcinoma;squamous cell carcinoma; lymphoepithelial carcinoma; basal cellcarcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillarytransitional cell carcinoma; adenocarcinoma; gastrinoma, malignant;cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellularcarcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoidcystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma,familial polyposis coli; solid carcinoma; carcinoid tumor, malignant;branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma;chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma;basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma;follicular adenocarcinoma; papillary and follicular adenocarcinoma;nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma;endometroid carcinoma; skin appendage carcinoma; apocrineadenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma;mucoepidermoid carcinoma; cystadenocarcinoma; papillarycystadenocarcinoma; papillary serous cystadenocarcinoma; mucinouscystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma;infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma;inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma;adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma,malignant; ovarian stromal tumor, malignant; thecoma, malignant;granulosa cell tumor, malignant; and roblastoma, malignant; sertoli cellcarcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant;paraganglioma, malignant; extra-mammary paraganglioma, malignant;pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanoticmelanoma; superficial spreading melanoma; malig melanoma in giantpigmented nevus; epithelioid cell melanoma; blue nevus, malignant;sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma;liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonalrhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixedtumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma;carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant;phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant;dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii,malignant; choriocarcinoma; mesonephroma, malignant; hemangio sarcoma;hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma,malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma;chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma;giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant;ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblasticfibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant;ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillaryastrocytoma; astroblastoma; glioblastoma; oligodendroglioma;oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma;ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactoryneurogenic tumor; meningioma, malignant; neurofibrosarcoma;neurilemmoma, malignant; granular cell tumor, malignant; malignantlymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma;malignant lymphoma, small lymphocytic; malignant lymphoma, large cell,diffuse; malignant lymphoma, follicular; mycosis fungoides; otherspecified non-Hodgkin's lymphomas; malignant histiocytosis; multiplemyeloma; mast cell sarcoma; immunoproliferative small intestinaldisease; leukemia; lymphoid leukemia; plasma cell leukemia;erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia;basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mastcell leukemia; megakaryoblastic leukemia; myeloid sarcoma; plasmacytoma,colorectal cancer, rectal cancer, and hairy cell leukemia.

In some embodiments, the methods and compositions provided herein relateto the treatment of a leukemia. The term “leukemia” is meant broadlyprogressive, malignant diseases of the hematopoietic organs/systems andis generally characterized by a distorted proliferation and developmentof leukocytes and their precursors in the blood and bone marrow.Non-limiting examples of leukemia diseases include, acute nonlymphocyticleukemia, chronic lymphocytic leukemia, acute granulocytic leukemia,chronic granulocytic leukemia, acute promyelocytic leukemia, adultT-cell leukemia, aleukemic leukemia, a leukocythemic leukemia,basophilic leukemia, blast cell leukemia, bovine leukemia, chronicmyelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilicleukemia, Gross' leukemia, Rieder cell leukemia, Schilling's leukemia,stem cell leukemia, subleukemic leukemia, undifferentiated cellleukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblasticleukemia, histiocytic leukemia, stem cell leukemia, acute monocyticleukemia, leukopenic leukemia, lymphatic leukemia, lymphoblasticleukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoidleukemia, lymphosarcoma cell leukemia, mast cell leukemia,megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia,myeloblastic leukemia, myelocytic leukemia, myeloid granulocyticleukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cellleukemia, plasmacytic leukemia, and promyelocytic leukemia.

In some embodiments, the methods and compositions provided herein relateto the treatment of a carcinoma. The term “carcinoma” refers to amalignant growth made up of epithelial cells tending to infiltrate thesurrounding tissues, and/or resist physiological and non-physiologicalcell death signals and gives rise to metastases. Non-limiting exemplarytypes of carcinomas include, acinar carcinoma, acinous carcinoma,adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum,carcinoma of adrenal cortex, alveolar carcinoma, alveolar cellcarcinoma, basal cell carcinoma, carcinoma basocellulare, basaloidcarcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma,bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma,cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma,comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma encuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cellcarcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma,encephaloid carcinoma, epiennoid carcinoma, carcinoma epithelialeadenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum,gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma,signet-ring cell carcinoma, carcinoma simplex, small-cell carcinoma,solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma,carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma,string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes,transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma,verrucous carcinoma, carcinoma villosum, carcinoma gigantocellulare,glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma,hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma,hyaline carcinoma, hypernephroid carcinoma, infantile embryonalcarcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelialcarcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cellcarcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatouscarcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullarycarcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma,carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma,carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes,naspharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans,osteoid carcinoma, papillary carcinoma, periportal carcinoma,preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma,renal cell carcinoma of kidney, reserve cell carcinoma, carcinomasarcomatodes, schneiderian carcinoma, scirrhous carcinoma, merkel cellcarcinoma, salivary gland carcinoma and carcinoma scroti.

In some embodiments, the methods and compositions provided herein relateto the treatment of a sarcoma. The term “sarcoma” generally refers to atumor which is made up of a substance like the embryonic connectivetissue and is generally composed of closely packed cells embedded in afibrillar, heterogeneous, or homogeneous substance. Sarcomas include,but are not limited to, chondrosarcoma, fibrosarcoma, lymphosarcoma,melanosarcoma, myxosarcoma, osteosarcoma, endometrial sarcoma, stromalsarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giantcell sarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolarsoft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloromasarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma,granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmentedhemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma,immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma,Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymomasarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma,serocystic sarcoma, synovial sarcoma, and telangiectaltic sarcoma.

Additional exemplary neoplasias that can be treated using the methodsand compositions described herein include Hodgkin's Disease,Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, breast cancer,ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis,primary macroglobulinemia, small-cell lung tumors, primary brain tumors,stomach cancer, colon cancer, malignant pancreatic insulanoma, malignantcarcinoid, premalignant skin lesions, testicular cancer, lymphomas,thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tractcancer, malignant hypercalcemia, cervical cancer, endometrial cancer,and adrenal cortical cancer.

In some embodiments, the cancer treated is a melanoma. The term“melanoma” is taken to mean a tumor arising from the melanocytic systemof the skin and other organs. Non-limiting examples of melanomas areHarding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma,malignant melanoma, acral-lentiginous melanoma, amelanotic melanoma,benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, nodularmelanoma subungal melanoma, and superficial spreading melanoma.

Particular categories of tumors that can be treated using methods andcompositions described herein include lymphoproliferative disorders,breast cancer, ovarian cancer, prostate cancer, cervical cancer,endometrial cancer, bone cancer, liver cancer, stomach cancer, coloncancer, pancreatic cancer, cancer of the thyroid, head and neck cancer,cancer of the central nervous system, cancer of the peripheral nervoussystem, skin cancer, kidney cancer, as well as metastases of all theabove. Particular types of tumors include hepatocellular carcinoma,hepatoma, hepatoblastoma, rhabdomyosarcoma, esophageal carcinoma,thyroid carcinoma, ganglioblastoma, fibrosarcoma, myxosarcoma,liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, Ewing's tumor, leimyosarcoma, rhabdotheliosarcoma,invasive ductal carcinoma, papillary adenocarcinoma, melanoma, pulmonarysquamous cell carcinoma, basal cell carcinoma, adenocarcinoma (welldifferentiated, moderately differentiated, poorly differentiated orundifferentiated), bronchioloalveolar carcinoma, renal cell carcinoma,hypernephroma, hypernephroid adenocarcinoma, bile duct carcinoma,choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, testiculartumor, lung carcinoma including small cell, non-small and large celllung carcinoma, bladder carcinoma, glioma, astrocyoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, retinoblastoma, neuroblastoma,colon carcinoma, rectal carcinoma, hematopoietic malignancies includingall types of leukemia and lymphoma including: acute myelogenousleukemia, acute myelocytic leukemia, acute lymphocytic leukemia, chronicmyelogenous leukemia, chronic lymphocytic leukemia, mast cell leukemia,multiple myeloma, myeloid lymphoma, Hodgkin's lymphoma, non-Hodgkin'slymphoma.

Cancers treated in certain embodiments also include precancerouslesions, e.g., actinic keratosis (solar keratosis), moles (dysplasticnevi), acitinic chelitis (farmer's lip), cutaneous horns, Barrett'sesophagus, atrophic gastritis, dyskeratosis congenita, sideropenicdysphagia, lichen planus, oral submucous fibrosis, actinic (solar)elastosis and cervical dysplasia.

Cancers treated in some embodiments include non-cancerous or benigntumors, e.g., of endodermal, ectodermal or mesenchymal origin,including, but not limited to cholangioma, colonic polyp, adenoma,papilloma, cystadenoma, liver cell adenoma, hydatidiform mole, renaltubular adenoma, squamous cell papilloma, gastric polyp, hemangioma,osteoma, chondroma, lipoma, fibroma, lymphangioma, leiomyoma,rhabdomyoma, astrocytoma, nevus, meningioma, and ganglioneuroma.

EXAMPLES Example 1: Immune Modulation of Human Commensal Bacteria in aKLH-Based Delayed Type Hypersensitivity Model

Delayed-type hypersensitivity (DTH) is an animal model of atopicdermatitis (or allergic contact dermatitis), as reviewed by Petersen etal. (In vivo pharmacological disease models for psoriasis and atopicdermatitis in drug discovery. Basic & Clinical Pharm & Toxicology. 2006.99(2): 104-115; see also Irving C. Allen (ed.) Mouse Models of InnateImmunity: Methods and Protocols, Methods in Molecular Biology, 2013.vol. 1031, DOI 10.1007/978-1-62703-481-4_13). It can be induced in avariety of mouse and rat strains using various haptens or antigens, forexample an antigen emulsified with an adjuvant. DTH is characterized bysensitization as well as an antigen-specific T cell-mediated reactionthat results in erythema, edema, and cellular infiltration—especiallyinfiltration of antigen presenting cells (APCs), eosinophils, activatedCD4+ T cells, and cytokine-expressing Th2 cells.

The test formulations were prepared for KLH-based delayed typehypersensitivity model. The delayed-type hypersensitivity (DTH) modelprovides an in vivo mechanism to study the cell-mediated immuneresponse, and resulting inflammation, following exposure to a specificantigen to which the mice have been sensitized. Several variations ofthe DTH model have been used and are well known in the art (Irving C.Allen (ed.). Mouse Models of Innate Immunity: Methods and Protocols,Methods in Molecular Biology. Vol. 1031, DOI10.1007/978-1-62703-481-4_13, Springer Science+Business Media, LLC2013). For example, the emulsion of Keyhole Limpet Hemocyanin (KLH) andComplete Freund's Adjuvant (CFA) are prepared freshly on the day ofimmunization (day 0). To this end, 8 mg of KLH powder is weighed and isthoroughly re-suspended in 16 mL saline. An emulsion is prepared bymixing the KLH/saline with an equal volume of CFA solution (e.g. 10 mLKLH/saline+10 mL CFA solution) using syringes and a luer lock connector.KLH and CFA is mixed vigorously for several minutes to form awhite-colored emulsion to obtain maximum stability. A drop test isperformed to check if a homogenous emulsion is obtained, mixing iscontinued until an intact drop remains visible in the water.

On day 0, C57Bl/6J female mice, approximately 7 weeks old, were primedwith KLH antigen in CFA by subcutaneous immunization (4 sites, 50 μL persite).

Dexamethasone, a corticosteroid, is a known anti-inflammatory thatameliorates DTH reactions in mice, and serves as a positive control forsuppressing inflammation in this model (Taube and Carlsten, Action ofdexamethasone in the suppression of delayed-type hypersensitivity inreconstituted SCID mice. Inflamm Res. 2000. 49(10): 548-52). For thepositive control group, a stock solution of 17 mg/mL of Dexamethasonewas prepared on by diluting 6.8 mg Dexamethasone in 400 μL 96% ethanol.For each day of dosing, a working solution is prepared by diluting thestock solution 100× in sterile PBS to obtain a final concentration of0.17 mg/mL in a septum vial for intraperitoneal dosing.Dexamethasone-treated mice received 100 μL Dexamethasone i.p. (5 mL/kgof a 0.17 mg/mL solution). Frozen sucrose served as the negative control(vehicle). Lactococcus lactis cremoris Strain A was dosed at 100 ul ofbacterial cells at 1×10{circumflex over ( )}10 CFU/ml p.o. daily.Dexamethasone (positive control), vehicle (negative control), andLactococcus lactis cremoris Strain A were dosed daily.

On day 8, mice were challenged intradermally (i.d.) with 10 μg KLH insaline (in a volume of 10 μL) in the right ear and a control in the leftear Inflammatory response were measured using methods known in the art.Ear pinna thickness was measured at 48 hours following antigen challenge(FIGS. 1 and 3). As determined by ear thickness, Lactococcus lactiscremoris Strain A was as efficacious as Dexamethasone at suppressinginflammation compared to mice that received vehicle alone.

The efficacy of Lactococcus lactis cremoris Strain A may be studiedfurther using varied timing and varied doses. For instance, treatmentwith Lactococcus lactis cremoris Strain A-containing bacterialcomposition may be initiated at some point, either around the time ofpriming or around the time of DTH challenge. For example, Lactococcuslactis cremoris strain A (1×10⁹ CFU per mouse per day) may beadministered at the same time as the subcutaneous injections (day 0), orthey may be administered prior to, or upon, intradermal injection.Lactococcus lactis cremoris strain A is administered at varied doses andat defined intervals. For example, some mice are intravenously injectedwith Lactococcus lactis cremoris strain A at a range of between 1×10⁴and 5×10⁹ bacterial cells per mouse. While some mice will receiveLactococcus lactis cremoris strain A through i.v. injection, other micemay receive Lactococcus lactis cremoris strain A through intraperitoneal(i.p.) injection, subcutaneous (s.c.) injection, nasal routeadministration, oral gavage, topical administration, intradermal (i.d.)injection, or other means of administration. Some mice may receiveLactococcus lactis cremoris strain A every day (e.g. starting on day 0),while others may receive Lactococcus lactis cremoris strain A atalternative intervals (e.g. every other day, or once every three days).Additional groups of mice may receive some ratio of bacterial cells toLactococcus lactis cremoris strain A. The bacterial cells may be live,dead, or weakened. The bacterial cells may be harvested fresh (orfrozen) and administered, or they may be irradiated or heat-killed priorto administration.

For example, some groups of mice may receive between 1×10⁴ and 5×10⁹bacterial cells in an administration separate from, or comingled with,the Lactococcus lactis cremoris strain A administration. As with theLactococcus lactis cremoris strain A, bacterial cell administration maybe varied by route of administration, dose, and schedule. This caninclude oral gavage, i.v. injection, i.p. injection, i.d. injection,topical administration, or nasal route administration.

Some groups of mice may be treated with anti-inflammatory agent(s) (e.g.anti-CD154, blockade of members of the TNF family, or other treatment),and/or an appropriate control (e.g. vehicle or control antibody) atvarious time points and at effective doses.

In addition, some mice are treated with antibiotics prior to treatment.For example, vancomycin (0.5 g/L), ampicillin (1.0 g/L), gentamicin (1.0g/L) and amphotericin B (0.2 g/L) are added to the drinking water, andantibiotic treatment is halted at the time of treatment or a few daysprior to treatment. Some immunized mice are treated without receivingantibiotics.

Study animals may be sacrificed by exsanguination from the orbitalplexus under CO₂/O₂ anesthesia, followed by cervical dislocation on day10. For serum preparation, the blood samples are allowed to clot beforecentrifuging. The sera are transferred into clean tubes, each animal ina separate tube. Following exsanguination, of all animals both ears(each ear in a separate vial), the spleen, the mesenteric lymph nodes(MLN), the entire small intestine, and the colon are collected incryovials, snap frozen and stored at <−70° C.

Tissues may be dissociated using dissociation enzymes according to themanufacturer's instructions. Cells are stained for analysis by flowcytometry using techniques known in the art. Staining antibodies caninclude anti-CD11c (dendritic cells), anti-CD80, anti-CD86, anti-CD40,anti-MHCII, anti-CD8a, anti-CD4, and anti-CD103. Other markers that maybe analyzed include pan-immune cell marker CD45, T cell markers (CD3,CD4, CD8, CD25, Foxp3, T-bet, Gata3, Roryt, Granzyme B, CD69, PD-1,CTLA-4), and macrophage/myeloid markers (CD11b, MHCII, CD206, CD40,CSF1R, PD-L1, Gr-1, F4/80). In addition to immunophenotyping, serumcytokines are analyzed including, but not limited to, TNFa, IL-17,IL-13, IL-12p70, IL12p40, IL-10, IL-6, IL-5, IL-4, IL-2, IL-1b, IFNy,GM-CSF, G-CSF, M-CSF, MIG, IP10, MIP1b, RANTES, and MCP-1. Cytokineanalysis may be carried out on immune cells obtained from lymph nodes orother tissue, and/or on purified CD45+ infiltrated immune cells obtainedex vivo. Finally, immunohistochemistry is carried out on various tissuesections to measure T cells, macrophages, dendritic cells, andcheckpoint molecule protein expression.

Example 2: An Evaluation of Test Articles in the Modulation ofDSS-Induced Colitis in C57BL/6 Mice

Dextran sulfate sodium (DSS)-induced colitis is a well-studied animalmodel of colitis, as reviewed by Randhawa et al. (A review onchemical-induced inflammatory bowel disease models in rodents. Korean JPhysiol Pharmacol. 2014. 18(4): 279-288; see also Chassaing et al.Dextran sulfate sodium (DSS)-induced colitis in mice. Curr ProtocImmunol. 2014 Feb. 4; 104: Unit 15.25). In this model, mice are treatedwith DSS in drinking water, resulting in diarrhea and weight loss.

Groups of mice were treated with DSS to induce colitis as known in theart (Randhawa et al. 2014; Chassaing et al. 2014; see also Kim et al.Investigating intestinal inflammation in DSS-induced model of IBD. J VisExp. 2012. 60: 3678). For example, colitis was induced in mice byexposure to 3% DSS-treated drinking water from Day 0 to Day 5. One groupdid not receive DSS and served as naive controls. Animals were dosedwith sucrose vehicle (negative control), Lactococcus lactis cremorisStrain A (1×10⁹ CFU per mouse per day), Lactococcus lactis cremorisStrain X (1×10⁹ CFU per mouse per day), or anti-p40 positive control(administered i.p. on days 0, 3, 7, and 10). All animals were weigheddaily. As measured by decrease in weight loss, Lactococcus lactiscremoris Strain A was more efficacious than either anti-p40 (positivecontrol), or Bacteria A, B, or C (FIG. 2).

In other studies, treatment with Lactococcus lactis cremoris StrainA-containing bacterial composition may be initiated at some point,either on day 1 of DSS administration, or sometime thereafter. Forexample, Lactococcus lactis cremoris strain A may be administered at thesame time as DSS initiation (day 1), or they may be administered uponthe first signs of disease (e.g. weight loss or diarrhea), or during thestages of severe colitis. Mice may be observed daily for weight,morbidity, survival, presence of diarrhea and/or bloody stool.

Lactococcus lactis cremoris strain A is administered at varied doses,varied intervals, and/or varied routes of administration. For example,some mice are intravenously injected with Lactococcus lactis cremorisstrain A at a dose of between 1×10⁴ and 5×10⁹ bacterial cells per mouse.While some mice will receive Lactococcus lactis cremoris strain Athrough i.v. injection, other mice may receive Lactococcus lactiscremoris strain A through intraperitoneal (i.p.) injection, subcutaneous(s.c.) injection, nasal route administration, oral gavage, or othermeans of administration. Some mice may receive Lactococcus lactiscremoris strain A every day (e.g. starting on day 1), while others mayreceive Lactococcus lactis cremoris strain A at alternative intervals(e.g. every other day, or once every three days). Additional groups ofmice may receive some ratio of bacterial cells to Lactococcus lactiscremoris strain A. The bacterial cells may be live, dead, or weakened.The bacterial cells may be harvested fresh (or frozen) and administered,or they may be irradiated or heat-killed prior to administration.

Lactococcus lactis cremoris Strain A-containing bacterial compositionsmay be tested for their efficacy in a mouse model of DSS-inducedcolitis, either alone or in combination with whole bacterial cells, withor without the addition of other anti-inflammatory agents.

For example, some groups of mice may receive between 1×10⁴ and 5×10⁹bacterial cells in an administration separate from, or comingled with,the Lactococcus lactis cremoris strain A administration. As with theLactococcus lactis cremoris strain A, bacterial cell administration maybe varied by route of administration, dose, and schedule. This caninclude oral gavage, i.v. injection, i.p. injection, or nasal routeadministration.

Some groups of mice may be treated with additional anti-inflammatoryagent(s) (e.g. anti-CD154, blockade of members of the TNF family, orother treatment), and/or an appropriate control (e.g. vehicle or controlantibody) at various time points and at effective doses.

In addition, some mice are treated with antibiotics prior to treatment.For example, vancomycin (0.5 g/L), ampicillin (1.0 g/L), gentamicin (1.0g/L) and amphotericin B (0.2 g/L) are added to the drinking water, andantibiotic treatment is halted at the time of treatment or a few daysprior to treatment. Some mice receive DSS without receiving antibioticsbeforehand.

At various time points, mice undergo video endoscopy using a smallanimal endoscope (Karl Storz Endoskipe, Germany) under isofluraneanesthesia. Still images and video will be recorded to evaluate theextent of colitis and the response to treatment. Colitis will be scoredusing criteria known in the art. Fecal material will be collected forstudy.

The gastrointestinal (GI) tract, lymph nodes, and/or other tissues maybe removed for ex vivo histological, cytokine and/or flow cytometricanalysis using methods known in the art. For example, tissues areharvested and may be dissociated using dissociation enzymes according tothe manufacturer's instructions. Cells are stained for analysis by flowcytometry using techniques known in the art. Staining antibodies caninclude anti-CD11c (dendritic cells), anti-CD80, anti-CD86, anti-CD40,anti-MHCII, anti-CD8a, anti-CD4, and anti-CD103. Other markers that maybe analyzed include pan-immune cell marker CD45, T cell markers (CD3,CD4, CD8, CD25, Foxp3, T-bet, Gata3, Roryt, Granzyme B, CD69, PD-1,CTLA-4), and macrophage/myeloid markers (CD11b, MHCII, CD206, CD40,CSF1R, PD-L1, Gr-1, F4/80). In addition to immunophenotyping, serumcytokines are analyzed including, but not limited to, TNFa, IL-17,IL-13, IL-12p70, IL12p40, IL-10, IL-6, IL-5, IL-4, IL-2, IL-1b, IFNy,GM-CSF, G-CSF, M-CSF, MIG, IP10, MIP1b, RANTES, and MCP-1. Cytokineanalysis may be carried out on immune cells obtained from lymph nodes orother tissue, and/or on purified CD45+ GI tract-infiltrated immune cellsobtained ex vivo. Finally, immunohistochemistry is carried out onvarious tissue sections to measure T cells, macrophages, dendriticcells, and checkpoint molecule protein expression.

In order to examine the impact and longevity of disease protection,rather than being sacrificed, some mice may be rechallenged with adisease trigger. Mice will be analyzed for susceptibility to colitisseverity following rechallenge.

Following sacrifice, the colon, small intestine, spleen, and mesentericlymph nodes may be collected from all animals, and blood collected foranalysis.

Example 3: Lactococcus lactis Cremoris Strain A and/or EVs Derived fromLactococcus lactis Cremoris Strain A in a Mouse Model of ExperimentalAutoimmune Encephalomyelitis (EAE)

EAE is a well-studied animal model of multiple sclerosis, as reviewed byConstantinescu et al. (Experimental autoimmune encephalomyelitis (EAE)as a model for multiple sclerosis (MS). Br J Pharmacol. 2011 October;164(4): 1079-1106). It can be induced in a variety of mouse and ratstrains using different myelin-associated peptides, by the adoptivetransfer of activated encephalitogenic T cells, or the use of TCRtransgenic mice susceptible to EAE, as discussed in Mangalam et al. (Twodiscreet subsets of CD8+ T cells modulate PLP₉₁₋₁₁₀ induced experimentalautoimmune encephalomyelitis in HLA-DR3 transgenic mice. J Autoimmun.2012 June; 38(4): 344-353).

Lactococcus lactis cremoris Strain A-containing bacterial compositionsand/or EVs derived from Lactococcus lactis cremoris Strain A are testedfor their efficacy in the rodent model of EAE, either alone or incombination with whole bacterial cells, with or without the addition ofother anti-inflammatory treatments. For example, female 6-8 week oldC57Bl/6 mice are obtained from Taconic (Germantown, N.Y.). Groups ofmice will be administered two subcutaneous (s.c.) injections at twosites on the back (upper and lower) of 0.1 ml myelin oligodentrocyteglycoprotein 35-55 (MOG35-55; 100 ug per injection; 200 ug per mouse(total 0.2 ml per mouse)), emulsified in Complete Freund's Adjuvant(CFA; 2-5 mg killed Mycobacterium tuberculosis H37Ra/ml emulsion).Approximately 1-2 hours after the above, mice are intraperitoneally(i.p.) injected with 200 ng Pertussis toxin (PTx) in 0.1 ml PBS (2ug/ml). An additional IP injection of PTx is administered on day 2.Alternatively, an appropriate amount of an alternative myelin peptide(e.g. proteolipid protein (PLP)) will be used to induce EAE. Someanimals will serve as naïve controls. EAE severity will be assessed anda disability score will be assigned daily beginning on day 4 accordingto methods known in the art (Mangalam et al. 2012).

Treatment with Lactococcus lactis cremoris Strain A-containing bacterialcomposition and/or EVs derived from Lactococcus lactis cremoris Strain Ais initiated at some point, either around the time of immunization orfollowing EAE immunization. For example, Lactococcus lactis cremorisStrain A-containing bacterial and/or EVs derived from Lactococcus lactiscremoris Strain A composition may be administered at the same time asimmunization (day 1), or they may be administered upon the first signsof disability (e.g. limp tail), or during severe EAE. Lactococcus lactiscremoris Strain A-containing bacterial compositions and/or EVs derivedfrom Lactococcus lactis cremoris Strain A are administered at varieddoses and at defined intervals. For example, some mice are intravenouslyinjected with effective doses of Lactococcus lactis cremoris Strain A.For example, mice may receive between 1×10⁴ and 5×10⁹ bacterial cellsper mouse. While some mice will receive Lactococcus lactis cremorisstrain A and/or EVs derived from Lactococcus lactis cremoris Strain Athrough i.v. injection, other mice may receive Lactococcus lactiscremoris and/or EVs derived from Lactococcus lactis cremoris Strain Athrough intraperitoneal (i.p.) injection, subcutaneous (s.c.) injection,nasal route administration, oral gavage, or other means ofadministration. Some mice may receive Lactococcus lactis cremoris strainA and/or EVs derived from Lactococcus lactis cremoris Strain A every day(e.g. starting on day 1), while others may receive Lactococcus lactiscremoris strain A and/or EVs derived from Lactococcus lactis cremorisStrain A at alternative intervals (e.g. every other day, or once everythree days). Additional groups of mice may receive some ratio ofbacterial cells to Lactococcus lactis cremoris strain A. The bacterialcells may be live, dead, or weakened. The bacterial cells may beharvested fresh (or frozen) and administered, or they may be irradiatedor heat-killed prior to administration.

For example, some groups of mice may receive between 1×10⁴ and 5×10⁹bacterial cells in an administration separate from, or comingled with,the Lactococcus lactis cremoris strain A and/or EVs derived fromLactococcus lactis cremoris Strain A administration. As with theLactococcus lactis cremoris strain A, bacterial cell administration maybe varied by route of administration, dose, and schedule. This caninclude oral gavage, i.v. injection, i.p. injection, subcutaneous (s.c.)injection, or nasal route administration.

Some groups of mice may be treated with additional anti-inflammatoryagent(s) or EAE therapeutic(s) (e.g. anti-CD154, blockade of members ofthe TNF family, Vitamin D, or other treatment), and/or an appropriatecontrol (e.g. vehicle or control antibody) at various time points and ateffective doses.

In addition, some mice are treated with antibiotics prior to treatment.For example, vancomycin (0.5 g/L), ampicillin (1.0 g/L), gentamicin (1.0g/L) and amphotericin B (0.2 g/L) are added to the drinking water, andantibiotic treatment is halted at the time of treatment or a few daysprior to treatment. Some immunized mice are treated without receivingantibiotics.

At various time points, mice are sacrificed and sites of inflammation(e.g. brain and spinal cord), lymph nodes, or other tissues may beremoved for ex vivo histological, cytokine and/or flow cytometricanalysis using methods known in the art. For example, tissues aredissociated using dissociation enzymes according to the manufacturer'sinstructions. Cells are stained for analysis by flow cytometry usingtechniques known in the art. Staining antibodies can include anti-CD11c(dendritic cells), anti-CD80, anti-CD86, anti-CD40, anti-MHCII,anti-CD8a, anti-CD4, and anti-CD103. Other markers that may be analyzedinclude pan-immune cell marker CD45, T cell markers (CD3, CD4, CD8,CD25, Foxp3, T-bet, Gata3, Roryt, Granzyme B, CD69, PD-1, CTLA-4), andmacrophage/myeloid markers (CD11b, MHCII, CD206, CD40, CSF1R, PD-L1,Gr-1, F4/80). In addition to immunophenotyping, serum cytokines areanalyzed including, but not limited to, TNFa, IL-17, IL-13, IL-12p70,IL12p40, IL-10, IL-6, IL-5, IL-4, IL-2, IL-1b, IFNy, GM-CSF, G-CSF,M-CSF, MIG, IP10, MIP1b, RANTES, and MCP-1. Cytokine analysis may becarried out on immune cells obtained from lymph nodes or other tissue,and/or on purified CD45+ central nervous system (CNS)-infiltrated immunecells obtained ex vivo. Finally, immunohistochemistry is carried out onvarious tissue sections to measure T cells, macrophages, dendriticcells, and checkpoint molecule protein expression.

In order to examine the impact and longevity of disease protection,rather than being sacrificed, some mice may be rechallenged with adisease trigger (e.g. activated encephalitogenic T cells or re-injectionof EAE-inducing peptides). Mice will be analyzed for susceptibility todisease and EAE severity following rechallenge.

Example 4: Lactococcus lactis Cremoris Strain A and/or EVs Derived fromLactococcus lactis Cremoris Strain A in a Mouse Model ofCollagen-Induced Arthritis (CIA)

Collagen-induced arthritis (CIA) is an animal model commonly used tostudy rheumatoid arthritis (RA), as described by Caplazi et al. (Mousemodels of rheumatoid arthritis. Veterinary Pathology. Sep. 1, 2015.52(5): 819-826) (see also Brand et al. Collagen-induced arthritis.Nature Protocols. 2007. 2: 1269-1275; Pietrosimone et al.Collagen-induced arthritis: a model for murine autoimmune arthritis. BioProtoc. 2015 Oct. 20; 5(20): e1626).

Among other versions of the CIA rodent model, one model involvesimmunizing HLA-DQ8 Tg mice with chick type II collagen as described byTaneja et al. (J. Immunology. 2007. 56: 69-78; see also Taneja et al. J.Immunology 2008. 181: 2869-2877; and Taneja et al. Arthritis Rheum.,2007. 56: 69-78). Purification of chick CII has been described by Tanejaet al. (Arthritis Rheum., 2007. 56: 69-78). Mice are monitored for CIAdisease onset and progression following immunization, and severity ofdisease is evaluated and “graded” as described by Wooley, J. Exp. Med.1981. 154: 688-700.

Mice are immunized for CIA induction and separated into varioustreatment groups. Lactococcus lactis cremoris Strain A-containingbacterial compositions and/or EVs derived from Lactococcus lactiscremoris Strain A are tested for their efficacy in CIA, either alone orin combination with whole bacterial cells, with or without the additionof other anti-inflammatory treatments.

Treatment with Lactococcus lactis cremoris Strain A-containing bacterialcomposition and/or EVs derived from Lactococcus lactis cremoris Strain Ais initiated either around the time of immunization with collagen orpost-immunization. For example, in some groups, Lactococcus lactiscremoris strain A and/or EVs derived from Lactococcus lactis cremorisStrain A may be administered at the same time as immunization (day 1),or Lactococcus lactis cremoris strain A and/or EVs derived fromLactococcus lactis cremoris Strain A may be administered upon firstsigns of disease, or upon the onset of severe symptoms. Lactococcuslactis cremoris strain A and/or EVs derived from Lactococcus lactiscremoris Strain A is administered at varied doses and at definedintervals.

For example, some mice are intravenously injected with Lactococcuslactis cremoris strain A at a dose of between 1×10⁴ and 5×10⁹ bacterialcells per mouse. While some mice will receive Lactococcus lactiscremoris strain A and/or EVs derived from Lactococcus lactis cremorisStrain A through i.v. injection, other groups of mice may receiveLactococcus lactis cremoris strain A through intraperitoneal (i.p.)injection, subcutaneous (s.c.) injection, nasal route administration,oral gavage, or other means of administration. Some mice may receiveLactococcus lactis cremoris strain A and/or EVs derived from Lactococcuslactis cremoris Strain A every day (e.g. starting on day 1), whileothers may receive Lactococcus lactis cremoris strain A and/or EVsderived from Lactococcus lactis cremoris Strain A at alternativeintervals (e.g. every other day, or once every three days). Additionalgroups of mice may receive some ratio of bacterial cells to Lactococcuslactis cremoris strain A and/or EVs derived from Lactococcus lactiscremoris Strain A. The bacterial cells may be live, dead, or weakened.The bacterial cells may be harvested fresh (or frozen) and administered,or they may be irradiated or heat-killed prior to administration.

For example, some groups of mice may receive between 1×10⁴ and 5×10⁹bacterial cells in an administration separate from, or comingled with,the Lactococcus lactis cremoris strain A and/or EVs derived fromLactococcus lactis cremoris Strain A administration. As with theLactococcus lactis cremoris strain A, bacterial cell administration maybe varied by route of administration, dose, and schedule. This caninclude oral gavage, i.v. injection, i.p. injection, subcutaneous (s.c.)injection, intradermal (i.d.) injection, or nasal route administration.

Some groups of mice may be treated with additional anti-inflammatoryagent(s) or CIA therapeutic(s) (e.g. anti-CD154, blockade of members ofthe TNF family, Vitamin D, or other treatment), and/or an appropriatecontrol (e.g. vehicle or control antibody) at various time points and ateffective doses.

In addition, some mice are treated with antibiotics prior to treatment.For example, vancomycin (0.5 g/L), ampicillin (1.0 g/L), gentamicin (1.0g/L) and amphotericin B (0.2 g/L) are added to the drinking water, andantibiotic treatment is halted at the time of treatment or a few daysprior to treatment. Some immunized mice are treated without receivingantibiotics.

At various time points, serum samples are obtained to assess levels ofanti-chick and anti-mouse CII IgG antibodies using a standard ELISA(Batsalova et al. Comparative analysis of collagen type II-specificimmune responses during development of collagen-induced arthritis in twoB10 mouse strains. Arthritis Res Ther. 2012. 14(6): R237). Also, somemice are sacrificed and sites of inflammation (e.g. synovium), lymphnodes, or other tissues may be removed for ex vivo histological,cytokine and/or flow cytometric analysis using methods known in the art.The synovium and synovial fluid will be analyzed for plasma cellinfiltration and the presence of antibodies using techniques known inthe art. In addition, tissues are dissociated using dissociation enzymesaccording to the manufacturer's instructions to examine the profiles ofthe cellular infiltrates. Cells are stained for analysis by flowcytometry using techniques known in the art. Staining antibodies caninclude anti-CD11c (dendritic cells), anti-CD80, anti-CD86, anti-CD40,anti-MHCII, anti-CD8a, anti-CD4, and anti-CD103. Other markers that maybe analyzed include pan-immune cell marker CD45, T cell markers (CD3,CD4, CD8, CD25, Foxp3, T-bet, Gata3, Roryt, Granzyme B, CD69, PD-1,CTLA-4), and macrophage/myeloid markers (CD11b, MHCII, CD206, CD40,CSF1R, PD-L1, Gr-1, F4/80). In addition to immunophenotyping, serumcytokines are analyzed including, but not limited to, TNFa, IL-17,IL-13, IL-12p70, IL12p40, IL-10, IL-6, IL-5, IL-4, IL-2, IL-1b, IFNy,GM-CSF, G-CSF, M-CSF, MIG, IP10, MIP1b, RANTES, and MCP-1. Cytokineanalysis may be carried out on immune cells obtained from lymph nodes orother tissue, and/or on purified CD45+ synovium-infiltrated immune cellsobtained ex vivo. Finally, immunohistochemistry is carried out onvarious tissue sections to measure T cells, macrophages, dendriticcells, and checkpoint molecule protein expression.

In order to examine the impact and longevity of disease protection,rather than being sacrificed, some mice may be rechallenged with adisease trigger (e.g. activated re-injection with CIA-inducingpeptides). Mice will be analyzed for susceptibility to disease and CIAseverity following rechallenge.

Example 5: Lactococcus lactis Cremoris Strain a and/or EVs Derived fromLactococcus lactis Cremoris Strain a in a Mouse Model of Type 1 Diabetes(T1D)

Type 1 diabetes (T1D) is an autoimmune disease in which the immunesystem targets the islets of Langerhans of the pancreas, therebydestroying the body's ability to produce insulin.

There are various models of animal models of T1D, as reviewed by Belleet al. (Mouse models for type 1 diabetes. Drug Discov Today Dis Models.2009; 6(2): 41-45; see also Aileen J F King. The use of animal models indiabetes research. Br J Pharmacol. 2012 June; 166(3): 877-894. There aremodels for chemically-induced T1D, pathogen-induced T1D, as well asmodels in which the mice spontaneously develop T1D.

Lactococcus lactis cremoris Strain A-containing bacterial compositionsand/or EVs derived from Lactococcus lactis cremoris Strain A are testedfor their efficacy in a mouse model of T1D, either alone or incombination with whole bacterial cells, with or without the addition ofother anti-inflammatory treatments.

Depending on the method of T1D induction and/or whether T1D developmentis spontaneous, treatment with Lactococcus lactis cremoris strain Aand/or EVs derived from Lactococcus lactis cremoris Strain A isinitiated at some point, either around the time of induction orfollowing induction, or prior to the onset (or upon the onset) ofspontaneously-occurring T1D. Lactococcus lactis cremoris strain A and/orEVs derived from Lactococcus lactis cremoris Strain A is administered atvaried doses and at defined intervals. For example, some mice areintravenously injected with Lactococcus lactis cremoris strain A at adose of between 1×10⁴ and 5×10⁹ bacterial cells per mouse. Other micemay receive 25, 50, or 100 mg of Lactococcus lactis cremoris strain Aand/or EVs derived from Lactococcus lactis cremoris Strain A per mouse.While some mice will receive Lactococcus lactis cremoris strain A and/orEVs derived from Lactococcus lactis cremoris Strain A through i.v.injection, other mice may receive Lactococcus lactis cremoris strain Athrough intraperitoneal (i.p.) injection, subcutaneous (s.c.) injection,nasal route administration, oral gavage, or other means ofadministration. Some mice may receive Lactococcus lactis cremoris strainA and/or EVs derived from Lactococcus lactis cremoris Strain A everyday, while others may receive Lactococcus lactis cremoris strain A atalternative intervals (e.g. every other day, or once every three days).Additional groups of mice may receive some ratio of bacterial cells toLactococcus lactis cremoris strain A and/or EVs derived from Lactococcuslactis cremoris Strain A. The bacterial cells may be live, dead, orweakened. The bacterial cells may be harvested fresh (or frozen) andadministered, or they may be irradiated or heat-killed prior toadministration.

For example, some groups of mice may receive between 1×10⁴ and 5×10⁹bacterial cells in an administration separate from, or comingled with,the Lactococcus lactis cremoris strain A and/or EVs derived fromLactococcus lactis cremoris Strain A administration. As with theLactococcus lactis cremoris strain A, bacterial cell administration maybe varied by route of administration, dose, and schedule. This caninclude oral gavage, i.v. injection, i.p. injection, or nasal routeadministration.

Some groups of mice may be treated with additional treatments and/or anappropriate control (e.g. vehicle or control antibody) at various timepoints and at effective doses.

In addition, some mice are treated with antibiotics prior to treatment.For example, vancomycin (0.5 g/L), ampicillin (1.0 g/L), gentamicin (1.0g/L) and amphotericin B (0.2 g/L) are added to the drinking water, andantibiotic treatment is halted at the time of treatment or a few daysprior to treatment. Some immunized mice are treated without receivingantibiotics.

Blood glucose is monitored biweekly prior to the start of theexperiment. At various time points thereafter, nonfasting blood glucoseis measured. At various time points, mice are sacrificed and site thepancreas, lymph nodes, or other tissues may be removed for ex vivohistological, cytokine and/or flow cytometric analysis using methodsknown in the art. For example, tissues are dissociated usingdissociation enzymes according to the manufacturer's instructions. Cellsare stained for analysis by flow cytometry using techniques known in theart. Staining antibodies can include anti-CD11c (dendritic cells),anti-CD80, anti-CD86, anti-CD40, anti-MHCII, anti-CD8a, anti-CD4, andanti-CD103. Other markers that may be analyzed include pan-immune cellmarker CD45, T cell markers (CD3, CD4, CD8, CD25, Foxp3, T-bet, Gata3,Roryt, Granzyme B, CD69, PD-1, CTLA-4), and macrophage/myeloid markers(CD11b, MHCII, CD206, CD40, CSF1R, PD-L1, Gr-1, F4/80). In addition toimmunophenotyping, serum cytokines are analyzed including, but notlimited to, TNFa, IL-17, IL-13, IL-12p70, IL12p40, IL-10, IL-6, IL-5,IL-4, IL-2, IL-1b, IFNy, GM-CSF, G-CSF, M-CSF, MIG, IP10, MIP1b, RANTES,and MCP-1. Cytokine analysis may be carried out on immune cells obtainedfrom lymph nodes or other tissue, and/or on purified tissue-infiltratingimmune cells obtained ex vivo. Finally, immunohistochemistry is carriedout on various tissue sections to measure T cells, macrophages,dendritic cells, and checkpoint molecule protein expression. Antibodyproduction may also be assessed by ELISA.

In order to examine the impact and longevity of disease protection,rather than being sacrificed, some mice may be rechallenged with adisease trigger, or assessed for susceptibility to relapse. Mice will beanalyzed for susceptibility to diabetes onset and severity followingrechallenge (or spontaneously-occurring relapse).

Example 6: Lactococcus lactis Cremoris Strain A and/or EVs Derived fromLactococcus lactis Cremoris Strain A in a Mouse Model of PrimarySclerosing Cholangitis (PSC)

Primary Sclerosing Cholangitis (PSC) is a chronic liver disease thatslowly damages the bile ducts and leads to end-stage cirrhosis. It isassociated with inflammatory bowel disease (IBD).

There are various animal models for PSC, as reviewed by Fickert et al.(Characterization of animal models for primary sclerosing cholangitis(PSC). J Hepatol. 2014 June 60(6): 1290-1303; see also Pollheimer andFickert. Animal models in primary biliary cirrhosis and primarysclerosing cholangitis. Clin Rev Allergy Immunol. 2015 June 48(2-3):207-17). Induction of disease in PSC models includes chemical induction(e.g. 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-inducedcholangitis), pathogen-induced (e.g. Cryptosporidium parvum),experimental biliary obstruction (e.g. common bile duct ligation(CBDL)), and transgenic mouse model of antigen-driven biliary injury(e.g. Ova-Bil transgenic mice). For example, bile duct ligation isperformed as described by Georgiev et al. (Characterization oftime-related changes after experimental bile duct ligation. Br J Surg.2008. 95(5): 646-56), or disease is induced by DCC exposure as describedby Fickert et al. (A new xenobiotic-induced mouse model of sclerosingcholangitis and biliary fibrosis. Am J Path. Vol 171(2): 525-536.

Lactococcus lactis cremoris Strain A-containing bacterial compositionsand/or EVs derived from Lactococcus lactis cremoris Strain A are testedfor their efficacy in a mouse model of PSC, either alone or incombination with whole bacterial cells, with or without the addition ofsome other therapeutic agent.

DCC-Induced Cholangitis

For example, 6-8 week old C57bl/6 mice are obtained from Taconic orother vendor. Mice are fed a 0.1% DCC-supplemented diet for variousdurations. Some groups will receive DCC-supplement food for 1 week,others for 4 weeks, others for 8 weeks. Some groups of mice may receivea DCC-supplemented diet for a length of time and then be allowed torecover, thereafter receiving a normal diet. These mice may be studiedfor their ability to recover from disease and/or their susceptibility torelapse upon subsequent exposure to DCC. Treatment with Lactococcuslactis cremoris strain A and/or EVs derived from Lactococcus lactiscremoris Strain A is initiated at some point, either around the time ofDCC-feeding or subsequent to initial exposure to DCC. For example,Lactococcus lactis cremoris strain A and/or EVs derived from Lactococcuslactis cremoris Strain A may be administered on day 1, or they may beadministered sometime thereafter. Lactococcus lactis cremoris strain Aand/or EVs derived from Lactococcus lactis cremoris Strain A isadministered at varied doses and at defined intervals. For example, somemice are intravenously injected with Lactococcus lactis cremoris strainA at a range between 1×10⁴ and 5×10⁹ bacterial cells per mouse. Othermice may receive 25, 50, 100 mg of Lactococcus lactis cremoris strain Aper mouse. While some mice will receive Lactococcus lactis cremorisstrain A and/or EVs derived from Lactococcus lactis cremoris Strain Athrough i.v. injection, other mice may receive Lactococcus lactiscremoris strain A through i.p. injection, subcutaneous (s.c.) injection,nasal route administration, oral gavage, or other means ofadministration. Some mice may receive Lactococcus lactis cremoris strainA and/or EVs derived from Lactococcus lactis cremoris Strain A every day(e.g. starting on day 1), while others may receive Lactococcus lactiscremoris strain A and/or EVs derived from Lactococcus lactis cremorisStrain A at alternative intervals (e.g. every other day, or once everythree days). Additional groups of mice may receive some ratio ofbacterial cells to Lactococcus lactis cremoris strain A and/or EVsderived from Lactococcus lactis cremoris Strain A. The bacterial cellsmay be live, dead, or weakened. The bacterial cells may be harvestedfresh (or frozen), and administered, or they may be irradiated orheat-killed prior to administration. For example, some groups of micemay receive between 1×10⁴ and 5×10⁹ bacterial cells in an administrationseparate from, or comingled with, the Lactococcus lactis cremoris strainA and/or EVs derived from Lactococcus lactis cremoris Strain Aadministration. As with Lactococcus lactis cremoris strain A and/or EVsderived from Lactococcus lactis cremoris Strain A, bacterial celladministration may be varied by route of administration, dose, andschedule. This can include oral gavage, i.v. injection, i.p. injection,or nasal route administration. Some groups of mice may be treated withadditional agents and/or an appropriate control (e.g. vehicle orantibody) at various time points and at effective doses.

In addition, some mice are treated with antibiotics prior to treatment.For example, vancomycin (0.5 g/L), ampicillin (1.0 g/L), gentamicin (1.0g/L) and amphotericin B (0.2 g/L) are added to the drinking water, andantibiotic treatment is halted at the time of treatment or a few daysprior to treatment. Some immunized mice are treated without receivingantibiotics. At various time points, serum samples are analyzed for ALT,AP, bilirubin, and serum bile acid (BA) levels.

At various time points, mice are sacrificed, body and liver weight arerecorded, and sites of inflammation (e.g. liver, small and largeintestine, spleen), lymph nodes, or other tissues may be removed for exvivo histolomorphological characterization, cytokine and/or flowcytometric analysis using methods known in the art (see Fickert et al.Characterization of animal models for primary sclerosing cholangitis(PSC)). J Hepatol. 2014. 60(6): 1290-1303). For example, bile ducts arestained for expression of ICAM-1, VCAM-1, MadCAM-1. Some tissues arestained for histological examination, while others are dissociated usingdissociation enzymes according to the manufacturer's instructions. Cellsare stained for analysis by flow cytometry using techniques known in theart. Staining antibodies can include anti-CD11c (dendritic cells),anti-CD80, anti-CD86, anti-CD40, anti-MHCII, anti-CD8a, anti-CD4, andanti-CD103. Other markers that may be analyzed include pan-immune cellmarker CD45, T cell markers (CD3, CD4, CD8, CD25, Foxp3, T-bet, Gata3,Roryt, Granzyme B, CD69, PD-1, CTLA-4), and macrophage/myeloid markers(CD11b, MHCII, CD206, CD40, CSF1R, PD-L1, Gr-1, F4/80), as well asadhesion molecule expression (ICAM-1, VCAM-1, MadCAM-1). In addition toimmunophenotyping, serum cytokines are analyzed including, but notlimited to, TNFa, IL-17, IL-13, IL-12p70, IL12p40, IL-10, IL-6, IL-5,IL-4, IL-2, IL-1b, IFNy, GM-CSF, G-CSF, M-CSF, MIG, IP10, MIP1b, RANTES,and MCP-1. Cytokine analysis may be carried out on immune cells obtainedfrom lymph nodes or other tissue, and/or on purified CD45+ bileduct-infiltrated immune cells obtained ex vivo.

Liver tissue is prepared for histological analysis, for example, usingSirius-red staining followed by quantification of the fibrotic area. Atthe end of the treatment, blood is collected for plasma analysis ofliver enzymes, for example, AST or ALT, and to determine Bilirubinlevels. The hepatic content of Hydroxyproline can be measured usingestablished protocols. Hepatic gene expression analysis of inflammationand fibrosis markers may be performed by qRT-PCR using validatedprimers. These markers may include, but are not limited to, MCP-1,alpha-SMA, Coll1a1, and TIMP. Metabolite measurements may be performedin plasma, tissue and fecal samples using established metabolomicsmethods. Finally, immunohistochemistry is carried out on liver sectionsto measure neutrophils, T cells, macrophages, dendritic cells, or otherimmune cell infiltrates.

In order to examine the impact and longevity of disease protection,rather than being sacrificed, some mice may be rechallenged with DCC ata later time. Mice will be analyzed for susceptibility to cholangitisand cholangitis severity following rechallenge.

BDL-Induced Cholangitis

Alternatively, Lactococcus lactis cremoris Strain A-containing bacterialcompositions and/or EVs derived from Lactococcus lactis cremoris StrainA are tested for their efficacy in BDL-induced cholangitis. For example,6-8 week old C57Bl/6J mice are obtained from Taconic or other vendor.After an acclimation period the mice are subjected to a surgicalprocedure to perform a bile duct ligation (BDL). Some control animalsreceive a sham surgery. The BDL procedure leads to liver injury,inflammation and fibrosis within 7-21 days.

Treatment with Lactococcus lactis cremoris strain A and/or EVs derivedfrom Lactococcus lactis cremoris Strain A is initiated at some point,either around the time of surgery or some time following the surgery.Lactococcus lactis cremoris strain A and/or EVs derived from Lactococcuslactis cremoris Strain A is administered at varied doses and at definedintervals. For example, some mice are intravenously injected withLactococcus lactis cremoris strain A at a range between 1×10⁴ and 5×10⁹bacterial cells per mouse. Other mice may receive 25, 50, or 100 mg ofLactococcus lactis cremoris strain A per mouse. While some mice willreceive Lactococcus lactis cremoris strain A and/or EVs derived fromLactococcus lactis cremoris Strain A through i.v. injection, other micemay receive Lactococcus lactis cremoris strain A and/or EVs derived fromLactococcus lactis cremoris Strain A through i.p. injection,subcutaneous (s.c.) injection, nasal route administration, oral gavage,or other means of administration. Some mice receive Lactococcus lactiscremoris strain A and/or EVs derived from Lactococcus lactis cremorisStrain A every day (e.g. starting on day 1), while others may receiveLactococcus lactis cremoris strain A and/or EVs derived from Lactococcuslactis cremoris Strain A at alternative intervals (e.g. every other day,or once every three days). Additional groups of mice may receive someratio of bacterial cells to Lactococcus lactis cremoris strain A and/orEVs derived from Lactococcus lactis cremoris Strain A. The bacterialcells may be live, dead, or weakened. They bacterial cells may beharvested fresh (or frozen), and administered, or they may be irradiatedor heat-killed prior to administration. For example, some groups of micemay receive between 1×10⁴ and 5×10⁹ bacterial cells in an administrationseparate from, or comingled with, the Lactococcus lactis cremoris strainA and/or EVs derived from Lactococcus lactis cremoris Strain Aadministration. As with Lactococcus lactis cremoris strain A and/or EVsderived from Lactococcus lactis cremoris Strain A, bacterial celladministration may be varied by route of administration, dose, andschedule. This can include oral gavage, i.v. injection, i.p. injection,or nasal route administration. Some groups of mice may be treated withadditional agents and/or an appropriate control (e.g. vehicle orantibody) at various time points and at effective doses.

In addition, some mice are treated with antibiotics prior to treatment.For example, vancomycin (0.5 g/L), ampicillin (1.0 g/L), gentamicin (1.0g/L) and amphotericin B (0.2 g/L) are added to the drinking water, andantibiotic treatment is halted at the time of treatment or a few daysprior to treatment. Some immunized mice are treated without receivingantibiotics. At various time points, serum samples are analyzed for ALT,AP, bilirubin, and serum bile acid (BA) levels.

At various time points, mice are sacrificed, body and liver weight arerecorded, and sites of inflammation (e.g. liver, small and largeintestine, spleen), lymph nodes, or other tissues may be removed for exvivo histolomorphological characterization, cytokine and/or flowcytometric analysis using methods known in the art (see Fickert et al.Characterization of animal models for primary sclerosing cholangitis(PSC)). J Hepatol. 2014. 60(6): 1290-1303). For example, bile ducts arestained for expression of ICAM-1, VCAM-1, MadCAM-1. Some tissues arestained for histological examination, while others are dissociated usingdissociation enzymes according to the manufacturer's instructions. Cellsare stained for analysis by flow cytometry using techniques known in theart. Staining antibodies can include anti-CD11c (dendritic cells),anti-CD80, anti-CD86, anti-CD40, anti-MHCII, anti-CD8a, anti-CD4, andanti-CD103. Other markers that may be analyzed include pan-immune cellmarker CD45, T cell markers (CD3, CD4, CD8, CD25, Foxp3, T-bet, Gata3,Roryt, Granzyme B, CD69, PD-1, CTLA-4), and macrophage/myeloid markers(CD11b, MHCII, CD206, CD40, CSF1R, PD-L1, Gr-1, F4/80), as well asadhesion molecule expression (ICAM-1, VCAM-1, MadCAM-1). In addition toimmunophenotyping, serum cytokines are analyzed including, but notlimited to, TNFa, IL-17, IL-13, IL-12p70, IL12p40, IL-10, IL-6, IL-5,IL-4, IL-2, IL-1b, IFNy, GM-CSF, G-CSF, M-CSF, MIG, IP10, MIP1b, RANTES,and MCP-1. Cytokine analysis may be carried out on immune cells obtainedfrom lymph nodes or other tissue, and/or on purified CD45+ bileduct-infiltrated immune cells obtained ex vivo.

Liver tissue is prepared for histological analysis, for example, usingSirius-red staining followed by quantification of the fibrotic area. Atthe end of the treatment, blood is collected for plasma analysis ofliver enzymes, for example, AST or ALT, and to determine Bilirubinlevels. The hepatic content of Hydroxyproline can be measured usingestablished protocols. Hepatic gene expression analysis of inflammationand fibrosis markers may be performed by qRT-PCR using validatedprimers. These markers may include, but are not limited to, MCP-1,alpha-SMA, Coll1a1, and TIMP. Metabolite measurements may be performedin plasma, tissue and fecal samples using established metabolomicsmethods. Finally, immunohistochemistry is carried out on liver sectionsto measure neutrophils, T cells, macrophages, dendritic cells, or otherimmune cell infiltrates.

In order to examine the impact and longevity of disease protection,rather than being sacrificed, some mice may be analyzed for recovery.

Example 7: Lactococcus lactis Cremoris Strain A and/or EVs Derived fromLactococcus lactis Cremoris Strain A in a Mouse Model of NonalcoholicSteatohepatitis (NASH)

Nonalcoholic Steatohepattiis (NASH) is a severe form of NonalcoholicFatty Liver Disease (NAFLD), where buildup of hepatic fat (steatosis)and inflammation lead to liver injury and hepatocyte cell death(ballooning).

There are various animal models of NASH, as reviewed by Ibrahim et al.(Animal models of nonalcoholic steatohepatitis: Eat, Delete, andInflame. Dig Dis Sci. 2016 May. 61(5): 1325-1336; see also Lau et al.Animal models of non-alcoholic fatty liver disease: current perspectivesand recent advances 2017 January 241(1): 36-44).

Lactococcus lactis cremoris Strain A-containing bacterial compositionsare tested for their efficacy in a mouse model of NASH, either alone orin combination with whole bacterial cells, with or without the additionof another therapeutic agent. For example, 8-10 week old C57Bl/6J mice,obtained from Taconic (Germantown, N.Y.), or other vendor, are placed ona methionine choline deficient (MCD) diet for a period of 4-8 weeksduring which NASH features will develop, including steatosis,inflammation, ballooning and fibrosis.

Treatment with Lactococcus lactis cremoris strain A and/or EVs derivedfrom Lactococcus lactis cremoris Strain A is initiated at some point,either at the beginning of the diet, or at some point following dietinitiation (for example, one week after). For example, Lactococcuslactis cremoris strain A and/or EVs derived from Lactococcus lactiscremoris Strain A may be administered starting in the same day as theinitiation of the MCD diet. Lactococcus lactis cremoris strain A and/orEVs derived from Lactococcus lactis cremoris Strain A is administered atvaried doses and at defined intervals. For example, some mice areintravenously injected with Lactococcus lactis cremoris strain A atdoses between 1×10⁴ and 5×10⁹ bacterial cells per mouse. Other mice mayreceive 25, 50, or 100 mg of Lactococcus lactis cremoris strain A and/orEVs derived from Lactococcus lactis cremoris Strain A per mouse. Whilesome mice will receive Lactococcus lactis cremoris strain A and/or EVsderived from Lactococcus lactis cremoris Strain A through i.v.injection, other mice may receive Lactococcus lactis cremoris strain Aand/or EVs derived from Lactococcus lactis cremoris Strain A throughintraperitoneal (i.p.) injection, subcutaneous (s.c.) injection, nasalroute administration, oral gavage, or other means of administration.Some mice may receive Lactococcus lactis cremoris strain A and/or EVsderived from Lactococcus lactis cremoris Strain A every day (e.g.starting on day 1), while others may receive Lactococcus lactis cremorisstrain A and/or EVs derived from Lactococcus lactis cremoris Strain A atalternative intervals (e.g. every other day, or once every three days).Additional groups of mice may receive some ratio of bacterial cells toLactococcus lactis cremoris strain A and/or EVs derived from Lactococcuslactis cremoris Strain A. The bacterial cells may be live, dead, orweakened. The bacterial cells may be harvested fresh (or frozen) andadministered, or they may be irradiated or heat-killed prior toadministration.

For example, some groups of mice may receive between 1×10⁴ and 5×10⁹bacterial cells in an administration separate from, or comingled with,the Lactococcus lactis cremoris strain A and/or EVs derived fromLactococcus lactis cremoris Strain A administration. As with theLactococcus lactis cremoris strain A and/or EVs derived from Lactococcuslactis cremoris Strain A, bacterial cell administration may be varied byroute of administration, dose, and schedule. This can include oralgavage, i.v. injection, i.p. injection, or nasal route administration.Some groups of mice may be treated with additional NASH therapeutic(s)(e.g., FXR agonists, PPAR agonists, CCR2/5 antagonists or othertreatment) and/or appropriate control at various time points andeffective doses.

At various time points and/or at the end of the treatment, mice aresacrificed and liver, intestine, blood, feces, or other tissues may beremoved for ex vivo histological, biochemical, molecular or cytokineand/or flow cytometry analysis using methods known in the art. Forexample, liver tissues are weighed and prepared for histologicalanalysis, which may comprise staining with H&E, Sirius Red, anddetermination of NASH activity score (NAS). At various time points,blood is collected for plasma analysis of liver enzymes, for example,AST or ALT, using standards assays. In addition, the hepatic content ofcholesterol, triglycerides, or fatty acid acids can be measured usingestablished protocols. Hepatic gene expression analysis of inflammation,fibrosis, steatosis, ER stress, or oxidative stress markers may beperformed by qRT-PCR using validated primers. These markers may include,but are not limited to, IL-6, MCP-1, alpha-SMA, Coll1a1, CHOP, and NRF2.Metabolite measurements may be performed in plasma, tissue and fecalsamples using established biochemical and mass-spectrometry-basedmetabolomics methods. Serum cytokines are analyzed including, but notlimited to, TNFa, IL-17, IL-13, IL-12p70, IL12p40, IL-10, IL-6, IL-5,IL-4, IL-2, IL-1b, IFNy, GM-CSF, G-CSF, M-CSF, MIG, IP10, MIP1b, RANTES,and MCP-1. Cytokine analysis may be carried out on immune cells obtainedfrom lymph nodes or other tissue, and/or on purified CD45+ bileduct-infiltrated immune cells obtained ex vivo. Finally,immunohistochemistry is carried out on liver or intestine sections tomeasure neutrophils, T cells, macrophages, dendritic cells, or otherimmune cell infiltrates.

In order to examine the impact and longevity of disease protection,rather than being sacrificed, some mice may be analyzed for recovery.

Example 8: Lactococcus lactis Cremoris Strain A and/or EVs Derived fromLactococcus lactis Cremoris Strain A in a Mouse Model of Psoriasis

Psoriasis is a T-cell-mediated chronic inflammatory skin disease.So-called “plaque-type” psoriasis is the most common form of psoriasisand is typified by dry scales, red plaques, and thickening of the skindue to infiltration of immune cells into the dermis and epidermis.Several animal models have contributed to the understanding of thisdisease, as reviewed by Gudjonsson et al. (Mouse models of psoriasis. JInvest Derm. 2007. 127: 1292-1308; see also van der Fits et al.Imiquimod-induced psoriasis-like skin inflammation in mice is mediatedvia the IL-23/IL-17 axis. J. Immunol. 2009 May 1. 182(9): 5836-45).

Psoriasis can be induced in a variety of mouse models, including thosethat use transgenic, knockout, or xenograft models, as well as topicalapplication of imiquimod (IMQ), a TLR7/8 ligand.

Lactococcus lactis cremoris Strain A-containing bacterial compositionsand/or EVs derived from Lactococcus lactis cremoris Strain A are testedfor their efficacy in the mouse model of psoriasis, either alone or incombination with whole bacterial cells, with or without the addition ofother anti-inflammatory treatments. For example, 6-8 week old C57Bl/6 orBalb/c mice are obtained from Taconic (Germantown, N.Y.), or othervendor. Mice are shaved on the back and the right ear. Groups of micereceive a daily topical dose of 62.5 mg of commercially available IMQcream (5%) (Aldara; 3M Pharmaceuticals). The dose is applied to theshaved areas for 5 or 6 consecutive days. At regular intervals, mice arescored for erythema, scaling, and thickening on a scale from 0 to 4, asdescribed by van der Fits et al. (2009). Mice are monitored for earthickness using a Mitutoyo micrometer.

Treatment with Lactococcus lactis cremoris strain A and/or EVs derivedfrom Lactococcus lactis cremoris Strain A is initiated at some point,either around the time of the first application of IMQ, or somethingthereafter. For example, Lactococcus lactis cremoris strain A and/or EVsderived from Lactococcus lactis cremoris Strain A may be administered atthe same time as the subcutaneous injections (day 0), or they may beadministered prior to, or upon, application. Lactococcus lactis cremorisstrain A and/or EVs derived from Lactococcus lactis cremoris Strain A isadministered at varied doses and at defined intervals. For example, somemice are intravenously injected with Lactococcus lactis cremoris strainA at a dose of between 1×10⁴ and 5×10⁹ bacterial cells per mouse. Othermice may receive 25, 50, or 100 mg of Lactococcus lactis cremoris strainA per mouse. While some mice will receive Lactococcus lactis cremorisstrain A and/or EVs derived from Lactococcus lactis cremoris Strain Athrough i.v. injection, other mice may receive Lactococcus lactiscremoris strain A and/or EVs derived from Lactococcus lactis cremorisStrain A through intraperitoneal (i.p.) injection, nasal routeadministration, oral gavage, topical administration, intradermal (i.d.)injection, subcutaneous (s.c.) injection, or other means ofadministration. Some mice may receive Lactococcus lactis cremoris strainA and/or EVs derived from Lactococcus lactis cremoris Strain A every day(e.g. starting on day 0), while others may receive Lactococcus lactiscremoris strain A and/or EVs derived from Lactococcus lactis cremorisStrain A at alternative intervals (e.g. every other day, or once everythree days). Additional groups of mice may receive some ratio ofbacterial cells to Lactococcus lactis cremoris strain A and/or EVsderived from Lactococcus lactis cremoris Strain A. The bacterial cellsmay be live, dead, or weakened. The bacterial cells may be harvestedfresh (or frozen) and administered, or they may be irradiated orheat-killed prior to administration.

For example, some groups of mice may receive between 1×10⁴ and 5×10⁹bacterial cells in an administration separate from, or comingled with,the Lactococcus lactis cremoris strain A and/or EVs derived fromLactococcus lactis cremoris Strain A administration. As with theLactococcus lactis cremoris strain A and/or EVs derived from Lactococcuslactis cremoris Strain A, bacterial cell administration may be varied byroute of administration, dose, and schedule. This can include oralgavage, i.v. injection, i.p. injection, i.d. injection, s.c. injection,topical administration, or nasal route administration.

Some groups of mice may be treated with anti-inflammatory agent(s) (e.g.anti-CD154, blockade of members of the TNF family, or other treatment),and/or an appropriate control (e.g. vehicle or control antibody) atvarious time points and at effective doses.

In addition, some mice are treated with antibiotics prior to treatment.For example, vancomycin (0.5 g/L), ampicillin (1.0 g/L), gentamicin (1.0g/L) and amphotericin B (0.2 g/L) are added to the drinking water, andantibiotic treatment is halted at the time of treatment or a few daysprior to treatment. Some immunized mice are treated without receivingantibiotics.

At various time points, samples from back and ear skin are taken forcryosection staining analysis using methods known in the art. Othergroups of mice are sacrificed and lymph nodes, spleen, mesenteric lymphnodes (MLN), the small intestine, colon, and other tissues may beremoved for histology studies, ex vivo histological, cytokine and/orflow cytometric analysis using methods known in the art. Some tissuesmay be dissociated using dissociation enzymes according to themanufacturer's instructions. Cryosection samples, tissue samples, orcells obtained ex vivo are stained for analysis by flow cytometry usingtechniques known in the art. Staining antibodies can include anti-CD11c(dendritic cells), anti-CD80, anti-CD86, anti-CD40, anti-MHCII,anti-CD8a, anti-CD4, and anti-CD103. Other markers that may be analyzedinclude pan-immune cell marker CD45, T cell markers (CD3, CD4, CD8,CD25, Foxp3, T-bet, Gata3, Roryt, Granzyme B, CD69, PD-1, CTLA-4), andmacrophage/myeloid markers (CD11b, MHCII, CD206, CD40, CSF1R, PD-L1,Gr-1, F4/80). In addition to immunophenotyping, serum cytokines areanalyzed including, but not limited to, TNFa, IL-17, IL-13, IL-12p70,IL12p40, IL-10, IL-6, IL-5, IL-4, IL-2, IL-1b, IFNy, GM-CSF, G-CSF,M-CSF, MIG, IP10, MIP1b, RANTES, and MCP-1. Cytokine analysis may becarried out on immune cells obtained from lymph nodes or other tissue,and/or on purified CD45+ skin-infiltrated immune cells obtained ex vivo.Finally, immunohistochemistry is carried out on various tissue sectionsto measure T cells, macrophages, dendritic cells, and checkpointmolecule protein expression.

In order to examine the impact and longevity of psoriasis protection,rather than being sacrificed, some mice may be studied to assessrecovery, or they may be rechallenged with IMQ. The groups ofrechallenged mice will be analyzed for susceptibility to psoriasis andseverity of response.

Example 9: A Study of the Safety, Tolerability and Efficacy ofLactococcus lactis Cremoris Strain A as an Oral Therapeutic for theTreatment of Mild to Moderate Psoriasis or Atopic Dermatitis

A single-center, Phase 1 clinical study in performed in whichpreliminary safety, tolerability, and pharmacodynamic effect ofLactococcus lactis cremoris strain A is determined in healthyparticipants and participants with mild to moderate psoriasis or atopicdermatitis, but who are otherwise well.

This is a randomized, placebo-controlled clinical study with doseescalations and dose expansions to assess preliminary safety,tolerability, and pharmacodynamic effect of Lactococcus lactis cremorisstrain A, and is participant and investigator blind, sponsor unblinded,with single and multiple ascending doses. This investigation provides anopportunity to gain pharmacodynamic information using a range of tissuebiopsies and composite clinical endpoints.

The study consists of six (6) cohorts and will test doses of Lactococcuslactis cremoris strain A versus placebo. The initial three (3) cohortsare in healthy volunteers and will establish the safety and tolerabilityof Lactococcus lactis cremoris strain A. Once this has been established,the safety and tolerability in participants with psoriasis or atopicdermatitis will be tested, alongside pharmacodynamic effects on thesystemic immune system and observation of any clinical effects.

The treatment arms are described in Table 7, and optional additionalcohorts may be added to include dose expansion studies.

TABLE 7 Arms and Interventions Cohort Arms Assigned Interventions 1 12healthy volunteers: Lactococcus lactis cremoris 8 on Lactococcus lactisstrain A is orally cremoris strain A, administered 4 on placebo Drug:placebo oral capsule Dose = 66 mg capsule, once daily for 15 days 2 12healthy volunteers: 8 on Lactococcus lactis Lactococcus lactis cremoriscremoris strain A, strain A is orally 4 on placebo administered Dose =660 mg capsule, once Drug: placebo oral capsule daily for 15 days 3 12healthy volunteers: Lactococcus lactis cremoris 8 on Lactococcus lactisstrain A is orally cremoris strain A, administered 4 on placebo Drug:placebo oral capsule Dose = 3.3 g capsule, once daily for 15 days 4 12subjects with mild to Lactococcus lactis cremoris moderate psoriasis:strain A is orally 8 on Lactococcus lactis administered cremoris strainA, Drug: placebo oral capsule 4 on placebo Dose = 660 mg, capsule, oncedaily, 29 days 5 24 subjects with mild to Lactococcus lactis cremorismoderate psoriasis: strain A is orally 16 on Lactococcus lactisadministered cremoris strain A, Drug: placebo oral capsule 8 on placeboDose = 3.3 g, capsule, once daily, 29 days 6 24 subjects with mild toLactococcus lactis cremoris moderate atopic dermatitis: strain A isorally 16 on Lactococcus lactis administered cremoris strain A, Drug:placebo oral capsule 8 on placebo Dose = 3.3 g capsule, once daily, 29days

The study has at least three (3) outcome measures: 1) safety andtolerability; 2) clinical improvement in subjects with mild to moderatepsoriasis; and 3) clinical improvement in subjects with mild to moderateatopic dermatitis.

For (1) Safety and tolerability, serious adverse events (SAE), labmeasurements, electrocardiogram (ECG) measurements, vital signmeasurements, physical examination, Bristol stool scale, markers of GIintegrity, and immune biomarkers are conducted and assessed; for (2)Clinical improvement in subjects with mild to moderate psoriasis,psoriasis activity scoring index (PASI), investigators' globalassessment (IGA), and lesion severity score (LSS) are assessed over aperiod of 14 months; and for (3) Clinical improvement in subjects withmild to moderate atopic dermatitis, EASI, severity scoring of atopicdermatitis (SCORAD), LSS, and IGA are assessed over a period of 14months.

Anti-psoriasis and anti-atopic dermatitis activities are assessed by theInvestigator according to disease specific response criteria anddescribed in terms of objective response rate, duration of response,progression-free time-periods, clinical benefit rate, and diseasecontrol rate. Investigators will look for improvement from baseline ator around day 28 of dosing using the PASI and eczema activity scoringindex (EASI), both of which are known in the art.

Inclusion and Exclusion Criteria:

The inclusion criteria for all parts of the study include the following:

-   -   1. Participant has a body mass index of ≥18 kg/m2 to ≤35 kg/m2        at screening.    -   2. Participants who are overtly healthy as determined by medical        evaluation including medical history, physical examination,        laboratory tests, and cardia monitoring.    -   3. For patients with mild to moderate psoriasis:        -   a. Participant has had a confirmed diagnosis of mild to            moderate plaque-type psoriasis for at least 6 months            involving ≤5% of body surface area (BSA) (excluding the            scalp).        -   b. Participant has a minimum of 2 psoriatic lesions with at            least 1 plaque in a site suitable for biopsy.    -   4. For patients with mild to moderate atopic dermatitis:        -   a. Participant has mild to moderate atopic dermatitis with a            minimum of 3 to a maximum of 15% BSA involvement.        -   b. Participant has had a confirmed diagnosis of mild to            moderate atopic dermatitis for at least 6 months with IGA            score of 2 or 3.        -   c. Participant has a minimum of 2 atopic dermatitis lesions            with at least 1 in a site suitable for biopsy.

The following categories of patient are excluded from the study:

-   -   1. Female participant who is pregnant or plans to become        pregnant during the study, and/or female participant who is        breastfeeding or is sexually active with childbearing potential        who is not using a medically accepted birth control method.    -   2. Participant has received live attenuated vaccination within 6        weeks prior to screening or intends to have such a vaccination        during the course of the study.    -   3. Participant has received any investigational drug or        experimental procedure within 90 days or 5 half-lives, whichever        is longer, prior to study intervention administration.    -   4. Participant requires treatment with an anti-inflammatory drug        during the study period. Paracetamol will be permitted for use        as an antipyretic and/or analgesic (maximum of 2 grams/day in        any 24 hour period).    -   5. Participant has an active infection (e.g. sepsis, pneumonia,        abscess) or has had an infection requiring antibiotic treatment        within 6 weeks prior to investigational medicinal product (IMP)        administration. When in doubt, the investigator should confer        with the Sponsor study physician.    -   6. Participant has renal or liver impairment, defined as:        -   a. For healthy volunteers: i. for women, serum creatinine            level ≥125 μmol/L; for men, ≥135 μmol/L, or ii: Alanine            aminotransferase (ALT) and aspartate aminotransferase (AST)            ≥1.5× upper limit of normal (ULN), or iii. Alkaline            phosphatase (ALP) and/or bilirubin >1.5×ULN.        -   b. For participants with mild to moderate atopic dermatitis,            or psoriasis: i. For women, serum creatinine level ≥125            μmol/L; for men ≥135 μmol/L, or ii. ALT or AST>2×ULN and/or            bilirubin >1.5×ULN.

Dose Escalation Study

Patients receive all Lactococcus lactis cremoris strain A doses duringthe treatment period, or have had a dose-limiting toxicity (DLT) withinthe treatment period, may be considered evaluable for dose escalationdecisions. Dose escalation decisions occur when the cohort of patientshas met these criteria.

A DLT is defined as an adverse event (AE) or abnormal laboratory valuethat occurs within the first 7 days of treatment with Lactococcus lactiscremoris strain A, except for those that are clearly andincontrovertibly due to underlying disease, disease progression, orextraneous causes. Dose escalation decisions occur when the cohort ofpatients has met these criteria.

To implement dose escalation decisions, the available toxicityinformation (i.e., all AEs and all laboratory abnormalities regardlessof DLT assessment) is evaluated by the enrolling Investigators andSponsor medical monitor at a dose decision meeting or teleconference.Decisions are based on an evaluation of all relevant data available fromall dose cohorts evaluated in the ongoing study. Drug administration atthe next higher dose cohort may not proceed until the Investigatorreceives written confirmation from Sponsor indicating that the resultsof the previous dose cohort were evaluated and that it is permissible toproceed to the next higher dose cohort.

Intra-patient dose escalations are permitted for all cohorts after theintended dose level has been shown to be safe (i.e., all patientstreated at the intended dose level completed DLT assessments and ≤1patient experienced a DLT).

Example 10—Evaluation of Gene Deletion in Lactococcus lactis CremorisStrains in a KLH-Based Delayed Type Hypersensitivity Model

The efficacy of L. lactis cremoris Strains that lacked certain plasmidswas evaluated. Knockout strains were created using electroporationtechniques known in the art. Briefly, electrocompetent cells wereprepared by growing strain overnight in M17 media (5 g Pancreatic digestof casein, 5 g soy peptone, 5 g beef extract, 2.5 g yeast extract, 0.5 gascorbic acid, 0.25 g MgSO4, 19 g Disodium-β-glycerophosphate per L)that included 1% glucose. 2 mL of overnight culture was inoculated with50 mL of M17 media and allowed to grow to an optical density at 600 nmof 0.5-0.7 (about 5-7 hrs). The culture was then cooled on ice for 10min. Cells were spun down for 15 min at 3000 g and resuspended inelectroporation buffer (0.5M Sucrose+10% glycerol) which was repeated 2more times. Cells were then resuspended in 500 μL of electroporationbuffer and separated into 100 μL aliquots and stored at −80° C. untilelectroporation.

Electroporation proceeded by defrosting cells on ice prior to transferto an electroporation cuvette. Cell were then electroporated at 1.2 kVfor in Lactococcus lactis cremoris Strain A and 2.5 kV for inLactococcus lactis cremoris Strain B. 900 μL of recovery solution(M17+0.5M(0.17 g)Sucrose+0.5%(15 μl)Glucose+20 mM(10 μl)MgCl2+0.2 mM(10μl)CaCl2 per mL) was then immediately added. The cells were then kept onice for 10 min. Electroporated cells were subcultuted 1:10 in M17 mediaand incubated for 20 min at 30° C. before diluting and plating. Cellswere then screened for plasmid loss by PCR.

To elucidate the effect of the strains without the plasmids, theLactococcus lactis cremoris Strains A and B (both with and withoutplasmids) were evaluated in the mouse model of DTH. As noted above inExample 1, mice were injected with KLH and CFA i.d at 4 locations alongthe back (50 ug per mouse of KLH prepared in a 1:1 ratio with CFA in atotal volume of 50 ul per site). Mice were dosed for 9 days with1×10{circumflex over ( )}9 viable cells per day as follows: 1) anaerobicPBS (vehicle); 2) Lactococcus lactis cremoris Strain A; 3) Lactococcuslactis cremoris Strain A minus a 13 kb plasmid; 4) Lactococcus lactiscremoris Strain B; 5) Lactococcus lactis cremoris Strains B minus a 30kb plasmid; and 6) Dexamethasone (positive control). At 24 hourspost-challenge, the removal of a 13 kb plasmid from Lactococcus lactiscremoris Strain A reduced the efficacy of the strain while the removalof a 30 kb plasmid from Lactococcus lactis cremoris Strain B improvedthe efficacy of the strain (FIG. 4).

The strains were then sequenced to determine the genes within the 13 kbplasmid from) Lactococcus lactis cremoris Strain A and the 30 kb plasmidfrom Lactococcus lactis cremoris Strain B. See Table 5 and Table 6.

Example 11: Manufacturing Conditions

Enriched media is used to grow and prepare the bacterium for in vitroand in vivo use. For example, media may contain sugar, yeast extracts,plant based peptones, buffers, salts, trace elements, surfactants,anti-foaming agents, and vitamins. Composition of complex componentssuch as yeast extracts and peptones may be undefined or partiallydefined (including approximate concentrations of amino acids, sugarsetc.). Microbial metabolism may be dependent on the availability ofresources such as carbon and nitrogen. Various sugars or other carbonsources may be tested. Alternatively, media may be prepared and theselected bacterium grown as shown by Saarela et al., J. AppliedMicrobiology. 2005. 99: 1330-1339, which is hereby incorporated byreference. Influence of fermentation time, cryoprotectant andneutralization of cell concentrate on freeze-drying survival, storagestability, and acid and bile exposure of the selected bacterium producedwithout milk-based ingredients.

At large scale, the media is sterilized. Sterilization may be by UltraHigh Temperature (UHT) processing. The UHT processing is performed atvery high temperature for short periods of time. The UHT range may befrom 135-180° C. For example, the medium may be sterilized from between10 to 30 seconds at 135° C.

Inoculum can be prepared in flasks or in smaller bioreactors and growthis monitored. For example, the inoculum size may be betweenapproximately 0.5 and 3% of the total bioreactor volume. Depending onthe application and need for material, bioreactor volume can be at least2 L, 10 L, 80 L, 100 L, 250 L, 1000 L, 2500 L, 5000 L, 10,000 L.

Before the inoculation, the bioreactor is prepared with medium atdesired pH, temperature, and oxygen concentration. The initial pH of theculture medium may be different that the process set-point. pH stressmay be detrimental at low cell centration; the initial pH could bebetween pH 7.5 and the process set-point. For example, pH may be setbetween 4.5 and 8.0. During the fermentation, the pH can be controlledthrough the use of sodium hydroxide, potassium hydroxide, or ammoniumhydroxide. The temperature may be controlled from 25° C. to 45° C., forexample at 37° C. Anaerobic conditions are created by reducing the levelof oxygen in the culture broth from around 8 mg/L to 0 mg/L. Forexample, nitrogen or gas mixtures (N2, CO2, and H2) may be used in orderto establish anaerobic conditions. Alternatively, no gases are used andanaerobic conditions are established by cells consuming remaining oxygenfrom the medium. Depending on strain and inoculum size, the bioreactorfermentation time can vary. For example, fermentation time can vary fromapproximately 5 hours to 48 hours.

Reviving microbes from a frozen state may require specialconsiderations. Production medium may stress cells after a thaw; aspecific thaw medium may be required to consistently start a seed trainfrom thawed material. The kinetics of transfer or passage of seedmaterial to fresh medium, for the purposes of increasing the seed volumeor maintaining the microbial growth state, may be influenced by thecurrent state of the microbes (ex. exponential growth, stationarygrowth, unstressed, stressed).

Inoculation of the production fermenter(s) can impact growth kineticsand cellular activity. The initial state of the bioreactor system mustbe optimized to facilitate successful and consistent production. Thefraction of seed culture to total medium (e.g. a percentage) has adramatic impact on growth kinetics. The range may be 1-5% of thefermenter's working volume. The initial pH of the culture medium may bedifferent from the process set-point. pH stress may be detrimental atlow cell concentration; the initial pH may be between pH 7.5 and theprocess set-point. Agitation and gas flow into the system duringinoculation may be different from the process set-points. Physical andchemical stresses due to both conditions may be detrimental at low cellconcentration.

Process conditions and control settings may influence the kinetics ofmicrobial growth and cellular activity. Shifts in process conditions maychange membrane composition, production of metabolites, growth rate,cellular stress, etc. Optimal temperature range for growth may vary withstrain. The range may be 20-40° C. Optimal pH for cell growth andperformance of downstream activity may vary with strain. The range maybe pH 5-8. Gasses dissolved in the medium may be used by cells formetabolism. Adjusting concentrations of O₂, CO₂, and N₂ throughout theprocess may be required. Availability of nutrients may shift cellulargrowth. Microbes may have alternate kinetics when excess nutrients areavailable.

The state of microbes at the end of a fermentation and during harvestingmay impact cell survival and activity. Microbes may be preconditionedshortly before harvest to better prepare them for the physical andchemical stresses involved in separation and downstream processing. Achange in temperature (often reducing to 20-5° C.) may reduce cellularmetabolism, slowing growth (and/or death) and physiological change whenremoved from the fermenter. Effectiveness of centrifugal concentrationmay be influenced by culture pH. Raising pH by 1-2 points can improveeffectiveness of concentration but can also be detrimental to cells.Microbes may be stressed shortly before harvest by increasing theconcentration of salts and/or sugars in the medium. Cells stressed inthis way may better survive freezing and lyophilization duringdownstream.

Separation methods and technology may impact how efficiently microbesare separated from the culture medium. Solids may be removed usingcentrifugation techniques. Effectiveness of centrifugal concentrationcan be influenced by culture pH or by the use of flocculating agents.Raising pH by 1-2 points may improve effectiveness of concentration butcan also be detrimental to cells. Microbes may be stressed shortlybefore harvest by increasing the concentration of salts and/or sugars inthe medium. Cells stressed in this way may better survive freezing andlyophilization during downstream. Additionally, Microbes may also beseparated via filtration. Filtration is superior to centrifugationtechniques for purification if the cells require excessive g-minutes tosuccessfully centrifuge. Excipients can be added before afterseparation. Excipients can be added for cryo protection or forprotection during lyophilization. Excipients can include, but are notlimited to, sucrose, trehalose, or lactose, and these may bealternatively mixed with buffer and anti-oxidants. Prior tolyophilization, droplets of cell pellets mixed with excipients aresubmerged in liquid nitrogen.

Harvesting can be performed by continuous centrifugation. Product may beresuspended with various excipients to a desired final concentration.Excipients can be added for cryo protection or for protection duringlyophilization. Excipients can include, but are not limited to, sucrose,trehalose, or lactose, and these may be alternatively mixed with bufferand anti-oxidants. Prior to lyophilization, droplets of cell pelletsmixed with excipients are submerged in liquid nitrogen.

Lyophilization of material, including live bacteria, begins with primarydrying. During the primary drying phase, the ice is removed. Here, avacuum is generated and an appropriate amount of heat is supplied to thematerial for the ice to sublime. During the secondary drying phase,product bound water molecules are removed. Here, the temperature israised higher than in the primary drying phase to break anyphysico-chemical interactions that have formed between the watermolecules and the product material. The pressure may also be loweredfurther to enhance desorption during this stage. After the freeze-dryingprocess is complete, the chamber may be filled with an inert gas, suchas nitrogen. The product may be sealed within the freeze dryer under dryconditions, preventing exposure to atmospheric water and contaminants.

Example 12: Adoptive Transfer Delayed-Type Hypersensitivity (AdDTH)Mouse Model

Briefly, mice were purchased from Jackson Labs and allowed to acclimatein the vivarium for 1 week prior to start of experiment. Mice are housed5 animals per cage, in individually ventilated cages with standardbedding and enrichment. Standard Purina rodent diet (5001) andautoclaved water is provided ad libitum and checked daily. Ventilatedcages are changed once weekly. Animal housing rooms undergoes a lightingcycle consisting of 12 hours on and 12 hours off. Floors, walls, andceilings are sanitized once a month and rooms maintain a humidity rangebetween 30%-70%, and a temperature range between 68-79 degreesFahrenheit. Animal health checks are done twice daily.

On day −1, recipient BALB/c mice were adoptively transferred with1×10{circumflex over ( )}8 DO11. TCR Tg lymphocytes (i.p.).

On day 0, mice were anesthetized with isoflurane (one at a time) andtheir back was shaved. Mice were injected subcutaneously at 4 sites onthe back with 50 μl of Ovalbumin in CFA emulsion (Hooke Labs catalog#EK-0301).

A dexamethasone stock solution (17 mg/ml) was created by resuspending6.8 mg of dexamethasone in 400 μl of 96% ethanol. For each day ofdosing, a working solution is prepared by diluting the stock solution100× in sterile PBS to obtain a final concentration of 0.17 mg/mL in aseptum vial for intraperitoneal dosing. Dexamethasone-treated micereceived 100 μL Dexamethasone i.p. (5 mL/kg of a 0.17 mg/mL solution).Frozen sucrose served as the negative control (vehicle). Lactococcuslactis cremoris Strain A was dosed at 100 ul of bacterial cells at1×10{circumflex over ( )}10 CFU/ml daily. Dexamethasone (positivecontrol), vehicle (negative control), and Lactococcus lactis cremorisStrain A were dosed daily.

On days 1-9 mice were orally gavaged (groups 1 and 3) or injectedintraperitoneally (i.p. group 2).

On day 8, after all mice were gavaged, each mouse was anesthetized withisoflurane and a baseline left ear measurement was obtained using Fowlercalipers. Then 10 μl of OVA323-339 (Invivogen) (dissolved in sterile PBSto a concentration of 1 mg/ml) was injected intradermally in the leftear. As shown in FIG. 5, Lactococcus lactis cremoris Strain A reducesantigen-specific ear swelling (ear thickness) compared to vehicle(negative control), and anti-inflammatory Dexamethasone (positivecontrol) in an OVA based adoptive transfer delayed-type hypersensitivity(AdDTH) Mouse Model.

On day 9, a 24-hour ear measurement was obtained using Fowler calipersand mice were euthanized and tissues like spleen, draining cervicallymph nodes and mesenteric lymph nodes were collected for ex vivoprocessing.

Single cell suspensions of tissues were prepared, counted and plated to200,000 cells/well and restimulated with LPS and PMA/Ionomycin for 48hours or with OVA323-339 peptide or left unstimulated for 72 hours.Supernatants were collected at the end of stimulations and used fordownstream MSD or Luminex analyses.

As shown in FIGS. 6A, 6B, and 6C, Lactococcus lactis cremoris Strain Areduces expression of IL-12p70 (FIG. 6A), IL-22 (FIG. 6B), and KC (FIG.6C) in an Adoptive Transfer Delayed-Type Hypersensitivity (AdDTH) MouseModel. Circle represents vehicle, square represents dexamethasone, andtriangle represents Lactococcus lactis cremoris Strain A.

Example 13: Imiquimod Mouse Model of Psoriasis

Imiquimod driven psoriasis model is a Th17 driven skin inflammationmodel. Mice develop flakiness of the skin and erythema which mimics someof the pathology associated with human psoriasis that is scored on ascale of 0-4. Additionally, an ear inflammation may be assessed similarto the DTH.

Briefly, mice were purchased from Taconic Labs and allowed to acclimatein the vivarium for 1 week prior to start of experiment. Mice are housed5 animals per cage, in individually ventilated cages with standardbedding and enrichment. Standard Purina rodent diet (5001) andautoclaved water is provided ad libitum and checked daily. Ventilatedcages are changed once weekly. Animal housing rooms undergoes a lightingcycle consisting of 12 hours on and 12 hours off. Floors, walls, andceilings are sanitized once a month and rooms maintain a humidity rangebetween 30%-70%, and a temperature range between 68-79 degreesFahrenheit. Animal health checks are done twice daily.

A dexamethasone stock solution (17 mg/ml) was created by resuspending6.8 mg of dexamethasone in 400 μl of 96% ethanol. For each day ofdosing, a working solution is prepared by diluting the stock solution100× in sterile PBS to obtain a final concentration of 0.17 mg/mL in aseptum vial for intraperitoneal dosing. Dexamethasone-treated micereceived 100 μL Dexamethasone i.p. (5 mL/kg of a 0.17 mg/mL solution).Frozen sucrose served as the negative control (vehicle). Lactococcuslactis cremoris Strain A was dosed at 100 ul of bacterial cells at1×10{circumflex over ( )}10 CFU/ml p.o. daily. Dexamethasone (positivecontrol), vehicle (negative control), and Lactococcus lactis cremorisStrain A were dosed daily.

On Day 0, the backs of mice were shaved and the depilated with Nair (˜25sec). The Nair is then wiped off and backs of mice washed with warmwater (2×).

On Days 1-7, Aldara (5% Imiquimod 62.5 mg per mouse) or control cream isapplied on the backs of mice. The cream is re-spread to ensure uniformapplication. Every day an inflammation skin score is recorded.

On Day 8, back skin punches are collected for downstream RNA analyses.Skin inflammation scores are evaluated based on the following scale:0—normal, no reaction; 1—slight erythema; 2—moderate to severe erythemaand some plaques; 3—marked erythema and plaques; 4—very marked erythemaand plaques. As depicted in FIG. 7, Lactococcus lactis cremoris Strain Aimproved the skin inflammation scores in an imiquimod model of psoriasiscompared to control cream, vehicle, and dexamethasone.

INCORPORATION BY REFERENCE

All publications patent applications mentioned herein are herebyincorporated by reference in their entirety as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference. In case of conflict, thepresent application, including any definitions herein, will control.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed is: 1-45. (canceled)
 46. A bacterial compositioncomprising Lactococcus lactis cremoris Strain A (ATCC Deposit NumberPTA-125368) and a pharmaceutically acceptable carrier. 47-49. (canceled)50. The bacterial composition of claim 46, wherein the bacterialcomposition formulated for oral, rectal, intravenous, intratumoral, orsubcutaneous administration.
 51. The bacterial composition of claim 46,wherein at least 50% of the bacteria in the bacterial composition areLactococcus lactis cremoris Strain A (ATCC Deposit Number PTA-125368).52. The bacterial composition of claim 46, wherein at least 90% of thebacteria in the bacterial composition are Lactococcus lactis cremorisStrain A (ATCC Deposit Number PTA-125368).
 53. The bacterial compositionof claim 46, wherein substantially all of the bacteria in the bacterialcomposition are the immune modulating Lactococcus strain.
 54. Thebacterial composition of claim 46, wherein the bacterial compositioncomprises at least 1×10⁶ colony forming units (CFUs) of Lactococcuslactis cremoris Strain A (ATCC Deposit Number PTA-125368).
 55. Thebacterial composition of claim 54, wherein the bacterial compositioncomprises at least 1×10⁷ CFUs of Lactococcus lactis cremoris Strain A(ATCC Deposit Number PTA-125368).
 56. The bacterial composition of claim54, wherein the bacterial composition comprises at least 1×10⁸ CFUs ofLactococcus lactis cremoris Strain A (ATCC Deposit Number PTA-125368).57-59. (canceled)
 60. The bacterial composition of claim 46, wherein thebacterial composition comprises live bacteria.
 61. The bacterialcomposition of claim 46, wherein the bacterial composition comprisesattenuated bacteria.
 62. The bacterial composition of claim 46, whereinthe bacterial composition comprises killed bacteria.
 63. The bacterialcomposition of claim 46, wherein the bacterial composition comprisesirradiated bacterium.
 64. The bacterial composition of claim 63, whereinthe bacterial composition comprises gamma irradiated bacterium.
 65. Thebacterial composition of claim 46, wherein administration of thebacterial composition treats an immune disorder.
 66. The bacterialcomposition of claim 46, wherein administration of the bacterialcomposition induces an immune response.
 67. The bacterial composition ofclaim 46, wherein the bacterial composition is formulated with anenteric coating or micro encapsulation. 68-79. (canceled)
 80. Thebacterial composition of claim 46, wherein the bacterial composition issubstantially free of exopolysaccharides. 81-94. (canceled)
 95. Thebacterial composition of claim 46, wherein the bacterial composition isformulated for administration in solid form.
 96. The bacterialcomposition of claim 46, wherein the bacterial composition is formulatedin a capsule.